Detection Of Aflatoxin M1 Research Articles

Biosensor based on antifouling PEG/Gold nanoparticles composite for sensitive detection of aflatoxin M1 in milk

Detection of ultra-trace amounts of aflatoxin M1 (AFM1) is an important requirement for food safety since it is a toxic mycotoxin, present in cow milk, with strictly regulated low levels. To achieve detection of low levels of AFM1, we developed a new nanometric aptasensing platform based on the modified SPCE, which was decorated with AuNPs, a tetraethylene glycol ferrocene derivative and an anti-AFM1 aptamer. The ferrocene tethered to AuNPs served as a capacitance transducer contributing to an interfacial charge. The capacitive signal was used to quantify the toxin. This study revealed a good sensitivity toward the toxin with a dynamic range of 20 to 300 pg·mL−1 and a LOD as low as 7.14 pg·mL−1 (S/N = 3). The nanoaptosensor exhibits very good metrological performances. To evaluate the sensing platform, the latter was applied to detect the presence of AFM1 in pasteurized cow milk.

An ultrasensitive, homogeneous fluorescence quenching immunoassay integrating separation and detection of aflatoxin M1 based on magnetic graphene composites.

Ahomogeneous fluorescence quenching immunoassay is describedfor simultaneous separation and detection of aflatoxin M1 (AFM1) in milk. The novel assay relies on monoclonal antibody (mAb) functionalized Fe3O4 decorated reduced-graphene oxide (rGO-Fe3O4-mAb) as both capture probe and energy acceptor, combined with tetramethylrhodamine cadaverine-labeled aflatoxin B1 (AFB1-TRCA) as the energy donor. In the assay, AFB1-TRCA binds to rGO-Fe3O4-mAb in the absence of AFM1, quenching the fluorescence of TRCA by resonance energy transfer. Significantly, the immunoassay integrates sample preparation and detection into a single step, by using magnetic graphene composites to avoid washing and centrifugation steps, and the assay can be completed within 10min. Under optimized conditions, the visual and quantitative detection limits of the assay for AFM1 were 50 and 3.8ngL-1, respectively, which were significantly lower than those obtained by fluorescence polarization immunoassay using the same immunoreagents. Owing to its operation and highly sensitivity, the proposed assay provides a powerful tool for the detection of AFM1.

Detection of aflatoxin M1 by fiber cavity attenuated phase shift spectroscopy.

Aflatoxin M1 (AFM1) is a carcinogenic compound commonly found in milk in excess of the WHO permissible limit, especially in developing countries. Currently, state-of-the-art tests for detecting AFM1 in milk include chromatographic systems and enzyme-linked-immunosorbent assays. Although these tests provide fair accuracy and sensitivity, they require trained laboratory personnel, expensive infrastructure, and many hours to produce final results. Optical sensors leveraging spectroscopy have a tremendous potential of providing an accurate, real-time, and specialist-free AFM1 detector. Despite this, AFM1 sensing demonstrations using optical spectroscopy are still immature. Here, we demonstrate an optical sensor that employs the principle of cavity attenuated phase shift spectroscopy in optical fiber cavities for rapid AFM1 detection in aqueous solutions at 1550 nm. The sensor constitutes a cavity built by two fiber Bragg gratings. We splice a tapered fiber of < 10 μm waist inside the cavity as a sensing head. For ensuring specific binding of AFM1 in a solution, the tapered fiber is functionalized with DNA aptamers followed by validation of the conjugation via FTIR, TGA, and EDX analyses. We then detect AFM1 in a solution by measuring the phase shift between a sinusoidally modulated laser input and the sensor output at resonant frequencies of the cavity. Our results show that the sensor has the detection limit of 20 ng/L (20 ppt), which is well below both the U.S. and the European safety regulations. We anticipate that the present work will lead towards a rapid and accurate AFM1 sensor, especially for low-resource settings.

Validation of I'screen AFLA M1 Milk for Detection of Aflatoxin M1 in Raw Bovine Milk and Powdered Milk: AOAC Performance Tested MethodSM 072002.

Aflatoxin M1 (AFM1) is the major metabolite of Aflatoxin B1 (AFB1) and can be found in the milk of animals fed with feed containing AFB1. The frequency of occurrence of AFM1 in milk has led to the development of specific quantitative methods of analysis to mitigate the risk of adversely affecting human health. To demonstrate that the I'screen AFLA M1 Milk ELISA kit can quantify AFM1 in raw bovine milk and powdered milk. Assay performance was evaluated studying lot-to-lot consistency, assay stability, robustness, and possible interferences of related molecules. Raw bovine milk samples spiked at 0, 5.0, 20, 50, 100, and 200 ng/L of AFM1 and powdered milk reference materials and spiked samples at 100 and 200 ng/L were tested to determine recovery, repeatability, and bias. LOD and LOQ were also determined for both matrices. High selectivity for AFM1 was demonstrated and performances were consistent, robust, and stable. The LOQ was validated at 5 ng/L for raw milk and 50 ng/L for powdered milk. Recoveries for spiked raw and powdered milk were 97-122%, with RSDr < 10%, and 106-111% for reference materials, with RSDr < 5%. The data collected validate the method as a selective, specific, sensitive, accurate, and precise tool for the analysis of AFM1 in raw bovine milk and powdered milk. We demonstrated that I'screen AFLA M1 is a reliable kit and a proper screening tool suitable for high analytical throughputs.

Development of electrochemical aptasensor based on gold nanoparticles and electrospun carbon nanofibers for the detection of aflatoxin M1 in milk

An electrochemical aptasensor was developed based on electrospun carbon nanofiber (ECNF) mat for detection of aflatoxin M1 (AFM1) in the milk samples. The ECNF mat was firstly produced by electrospinning and heat treatment method utilized directly as a novel substrate electrode. This electrode was then modified by electrodeposition of gold nanoparticles (AuNPs) and immobilization of a thiol-modified single stranded DNA (ss-HSDNA), respectively. The structure and morphology of prepared ECNF mat and AuNPs/ECNF mat electrode were characterized by means of Raman spectroscopy and scanning electron microscopy (SEM) equipped with energy dispersive spectroscopy (EDS). The characterization results indicated that the gold nanopartciles were uniformly electrodeposited on ECNF mat electrode. To analysis each step of the aptasensor development, cyclic voltammetry (CV) experiments were carried out in the [Fe(CN)6]−3/−4 solution. The biosensor exhibited a low detection limit (0.6 pg/mL), a wide and useful linear range (1-100 pg/mL), high sensitivity, excellent stability and reproducibility as well as good recovery. Moreover, the results obtained in this research were comparable to those obtained using the HPLC. Thus, good electrochemical performance and simple preparation procedure of the ss-HSDNA/AuNPs/ECNF mat electrode can provide promising potential for the detection of AFM1 in milk samples.

Determination of trace aflatoxin M1 (AFM1) residue in milk by an immunochromatographic assay based on (PEI/PSS)4 red silica nanoparticles.

Aflatoxin M1 (AFM1) residues in milk pose a major threat to human health, so there is an urgent need for a simple, rapid, and sensitive method for the determination of trace AFM1 in milk. In this study, a competitive immunochromatographic assay (ICA), using visual (PEI/PSS)4 red silica nanoparticles (SiNPs) as signal amplification probes, was used for the highly sensitive detection of AFM1. The (PEI/PSS)4 red SiNPs were used to label AFM1 monoclonal antibody (mAb) to prepare ICA for the detection of AFM1. After exploring the optimal conditions of mAb and immunoprobe dosage conditions, the lowest visual detection limit (VDL) of AFM1 in phosphate-buffered saline with Tween 20 (PBST, 10mM, pH7.4, containing 1% BSA, 3% sucrose, 1% trehalose, and 0.5% Tween 20) can reach 0.1pg/mL. The intuitive visually visible value of AFM1 in both PBST and milk was 10pg/mL. The results showed that the immunochromatographic system based on high chroma color (PEI/PSS)4 red SiNPs has high sensitivity and broad application prospects for the detection of trace AFM1 residues in milk. The high chroma (PEI/PSS)4 red SiNPs are expected to be a convenient biomarker for improving the sensitivity of immune chromatography bands. Graphical abstract The schematic diagram shows the detection principle. In this work, in the competitive experiment, (PEI/PSS)4 red SiNPs were selected as visual labeling materials, and the specific antibody combined with the labeled material was selected as an immune probe. The AFM1-BSA antigen coupled with the macromolecular BSA was fixed on the T line of the nitrocellulose (NC) membrane. The AFM1 in sample solution competes with AFM1-BSA for the specific binding site of immune probe. The detection sensitivity of this method for AFM1 is obtained by judging the change of the red signal intensity produced by the positive sample, compared with the color at the T line of the negative sample.

An optical aptasensor for aflatoxin M1 detection based on target-induced protection of gold nanoparticles against salt-induced aggregation and silica nanoparticles.

Not only intoxications of aflatoxins are significant risk for human beings, but also; the contamination with these toxins affect the economy. Therefore, developing a rapid, precise and inexpensive determination method is vitally important. Here, a colorimetric aptasensor is introduced for the detection of aflatoxin M1 (AFM1) based on the preservation of gold nanoparticles (AuNPs) against NaCl-induced aggregation by detaching of complementary strand of aptamer (CS) from the aptamer-modified streptavidin coated silica nanoparticles (SNPs) following the addition of target. So, the color of sample remains red. While, in the lack of AFM1, salt-induced aggregation of AuNPs occurs and the color of sample becomes purple as the aptamer/CS (dsDNA)-modified SNPs is stable and CS cannot bind to AuNPs. The proposed aptasensor could detect AFM1 in a linear dynamic range, 300-75,000ng/L, with a detection limit of 30ng/L. Also, the sensing method was effectively applied for AFM1 recognition in milk samples.

A Sensitive Two-Analyte Immunochromatographic Strip for Simultaneously Detecting Aflatoxin M1 and Chloramphenicol in Milk.

A two-analyte immunochromatographic strip (immunostrip) was developed for the simultaneous detection of aflatoxin M1 (AFM1) and chloramphenicol (CAP) in milk. Protein conjugates (AFM1-ovalbumin (OVA) and CAP-OVA) and goat anti-rabbit IgG were respectively drawn on nitrocellulose membrane as two test lines (T1 and T2) and a control line (C). The immunostrip was dipped into a well that contained a 200 μL milk sample, 5 μL AFM1 antibody-gold conjugates, and 8 μL CAP antibody-gold conjugates; the whole assay was completed in 15 min and the results could be interpreted visually or using a reader. This immunostrip has cut-off levels of 0.1 ng/mL and 0.5 ng/mL for AFM1 and CAP, respectively. Analysis of CAP and AFM1 in milk samples revealed that data from the immunostrip test agreed closely with those obtained from ELISA. The two-analyte immunostrip is a rapid way for on-site simultaneous detection of AFM1 and CAP in milk.

Using ELISA technique for the detection of aflatoxin M1 in thick cream

Using ELISA technique for the detection of aflatoxin M1 in thick cream

Evaluation and Assessment of Aflatoxin M1 in Milk and Milk Products in Yemen Using High-Performance Liquid Chromatography

Aflatoxin M1 is one of the major fungal contaminants found in dairy products around the globe. The objective of this study was to investigate the incidence and occurrence of aflatoxin M1 (AFM1) in samples of milk and milk products in Yemen. The tested dairy product samples were collected from different sources for aflatoxin M1 (AFM1) in Yemen. A total of 250 local and imported samples consisting of 38 liquid milk, 60 powder milk, 62 yogurt, and 90 cheese samples which are marketed throughout Yemen were tested by using high-performance liquid chromatography (HPLC) along with a fluorescence detector and immunoaffinity column purification for detection of AFM1. High levels of AFM1 were detected in preserved milk (77.24%), ranging from 0.021 μg/L to 5.95 μg/L. On the other hand, AFM1 was detected in 66.66% and 68.42% in powdered milk and liquid milk samples, respectively. Among dairy products, 87.09% of yogurt and 81.39% of cheese samples were found contaminated with AFM1. The AMF1 values were higher than the acceptable range for humans set by the European Union. So, we concluded that dairy products used in Yemen showed an AFM1 content beyond the acceptable range, and this is a major factor for causing health-related complications including cancer. In the present study, we reported for the first time the presence of mycotoxins especially AFM1 in dairy products used in Yemen.

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 and validation of a lateral flow immunoassay for the detection of aflatoxin M1 in raw and commercialised milks

Aflatoxin M1 (AFM1), a group I carcinogen, can be found in the milk of animals fed with aflatoxin‐B1‐contaminated feedstuff. In this study, we developed and validated a lateral flow immunoassay (LFIA) with the cut‐off value of 0.5 µg/L for AFM1 screening. The validation revealed that the assay yielded high sensitivity, selectivity and efficiency with no false positives. Forty‐one commercial milk samples were tested for AFM1 by the developed LFIA. However, the developed LFIA could not be applied to milk samples flavoured with chocolate and coffee. Otherwise, all samples gave negative test results.

Chemical composition and detection of Aflatoxin M1 in camels and cows milk in Sudan

ABSTRACT A hundred samples of milk from camels and cows were analysed for chemical composition and M1 contamination. The results indicated that camel milk from traditional nomadic systems revealed significantly (P0.05) higher values for protein, fat, lactose, solids-not-fat and density than that obtained from camel and cows kept in semi-intensive systems. Aflatoxin (AF) M1 was found in 33% of the milk samples with the highest occurrence (82.4%) in cow milk (35.3% ranged between 0.05 and 0.1 µ/kg and 47.1% ranged between 0.1 and 0.15 µ/kg) compared to milk samples from camel in semi-intensive systems (15.6% ranged between 0.05 and 0.1 µg/kg). All samples of milk from traditional nomadic systems revealed the absence of AFM1. These results revealed increasing of AF with increased fat content in milk, a decrease with decreasing other milk components, while the density of milk showed no relation.

Surface enhanced Raman scattering-based lateral flow immunosensor for sensitive detection of aflatoxin M1 in urine

Aflatoxin M1 (AFM1) is generally used as a biomarker in urine for the assessment of aflatoxin exposure in humans and animals. However, there is no approach for the rapid and on-site monitoring of AFM1 level in urine. Here, we report a surface enhanced Raman scattering (SERS)-based lateral flow immunosensor built for such a purpose. Raman molecule 5,5-dithiobis-2-nitrobenzoic acid and anti-AFM1 monoclonal antibody were conjugated with Au (core)@Ag (shell) nanoparticle to serve as SERS nanoprobe. AFM1-bovine serum albumin was conjugated with gold nanoparticle and then applied onto nitrocellulose membrane as a visible “GOLD” test line. Quantitation of AFM1 was performed by the readout of Raman signal from the SERS nanoprobes captured on the test line. After optimizing experimental parameters, the detection limit of this immunosensor can achieve as low as 1.7 pg/mL of AFM1 in urine, which is far below the recommended tolerable level (30 pg/mL) of AFM1 in urine. The spiking experiment yielded 93.8%–111.3% recovery with coefficients of variation below 17% demonstrating high assay accuracy and precision. Moreover, this immunosensing assay is fast with an assay time below 20 min. Therefore, the developed immunosensor is a promising tool for the rapid assessment of aflatoxin exposure in the field.

Determination of aflatoxin M1 in milk and milk powder using immuno-affinity column and fluorescence measurement

The method using immuno-affinity column clean-up and fluorescence measurement was evaluated for detection of aflatoxin M1 in milk and milk powder. Two kinds of commercial immuno-affinity columns used were Afla Test-P Column and AFLAPREP M. The results are shown below : (1) By using Afla Test-P Column and Fluorometer, the average recoveries of aflatoxin M1 from defatted milk spiked at the levels of 0.5 and 1.0ppb were 84.4±7.0% and 94.2±9.5%. The detection limit was 0.1ppb. This method is easy and suitable for multi-sample treatment. (2) By using AFLAPREP M, the average recoveries of aflatoxin M1 from defatted milk spiked at the levels of 0.1, 0.5 and 1.0ppb were 84.7±2.7 %, 88.7±1.9%and 89.2±4.2%. The detection limit was 0.05ppb. This method is more sensitive and can be used at the laboratory equipped with HPLC. (3) 25 samples of milk and 25 samples of milk powder were perchased from supermarkets in Taipei from July 1994 to June 1995 and tested by AFLAPREP M. Aflatoxin M1 was detected in 3 samples of milk. The toxin contents were 0.33, 0.12, 0.08ppb respectively. Milk powders were reconstituted and tested, no aflatoxin M1 was detected in 25 samples.

Gold nanorods etching-based plasmonic immunoassay for qualitative and quantitative detection of aflatoxin M1 in milk

In this study, we developed an indirect competitive plasmonic immunoassay using glucose oxidase (GOx)-induced redox reaction to etch Au nanorods (AuNRs) for qualitative and quantitative detection of aflatoxin M1 (AFM1) in milk. In this system, streptavidin (SA) was selected as a linker between biotinylated anti-AFM1-monoantibody (bio-mAb) and biotinylated GOx (bio-GOx) to form the immunocomplexes bio-mAb-SA-bio-GOx. After the oxidation of the glucose and I-, the resultant I2 could etch cetytrimethylammonium bromide (CTAB)-stabilized AuNRs. This resulted in the blue shift of the longitudinal localized surface plasmon resonance (LSPR) extinction peak, with a color change from blue to pink. The linear range and limit of detection (LOD) of the plasmonic immunoassay were 0.25–10 ng mL−1 and 0.11 ng mL−1, respectively. It displayed quantitative detection with high sensitivity, specificity, recovery, and accuracy, which is promising for qualitative and quantitative detection of AFM1 in food safety.

A novel fluorescent aptasensor for aflatoxin M1 detection using rolling circle amplification and g-C3N4 as fluorescence quencher

Aflatoxin M1 is a prevailing natural carcinogenic mycotoxin, found in milk. Herein, we reported a sensitive fluorescent aptasensor for AFM1, based on rolling circle amplification (RCA) technology to improve the sensitivity of the assay. The detection of AFM1 was achieved using KYF4: Eu3+ time-resolved fluorescent nanoparticles (KYF4: Eu3+ TRFNPs) as signal probe and graphitic carbon nitride (g-C3N4) nanosheet as a quencher. The AFM1 aptamer act as a recognition element and as a primer for DNA synthesis. TRFNPs attached to complementary DNA (TRFNPs-cDNA) acts as a signal probe. In the absence of target, a rolling circle template (RCT) ligated to aptamer and RCA reaction initiated. The TRFNPs-cDNA was strengthened to amplified RCA product (RCAP) to make RCAP/TRFNPs-cDNA duplex. This duplex cannot be adsorbed onto g-C3N4, and ultimately no fluorescence quenched. In the presence of AFM1, aptamer recognized the target and aptamer-target complex was formed. Hence, no aptamer hybridized with RCT, therefore, no RCA and duplex formation. Consequently, free TRFNPs-cDNA adsorbed onto g-C3N4 and resulted in quenched fluorescence. The assay provides a lower detection limit (0.0194 pg/mL) than the previously reported assays and has good recoveries between 92–99.8 %. Hence, the present method would be a suitable method for the quantification of small molecules.

Fabrication of microfluidic device for Aflatoxin M1 detection in milk samples with specific aptamers

This study describes the colorimetric detection of aflatoxin M1 (Afl M1) in milk samples using a microfluidic paper-based analytical device (µPAD). Fabrication of µPADs was done using a simple and quick approach. Each μPAD contained a detection zone and a sample zone interconnected by microchannels. The colorimetric assay was developed using unmodified AuNPs as a probe and 21-mer aptamer as a recognition molecule. The free aptamers were adsorbed onto the surface of AuNPs in absence of Afl M1, even at high salt concentrations. The salt induced aggregation of specific aptamers occurred in presence of Afl M1. Under optimum conditions, the analytical linear range was found to be 1 µM to 1 pM with limit of detection 3 pM and 10 nM in standard buffer and spiked milk samples respectively. The proposed aptamer based colorimetric assay was repeatable, quick, selective, and can be used for on-site detection of other toxins in milk and meat samples.

Detection Methods for Aflatoxin M1 in Dairy Products.

Mycotoxins are toxic compounds produced mainly by fungi of the genera Aspergillus, Fusarium and Penicillium. In the food chain, the original mycotoxin may be transformed in other toxic compounds, reaching the consumer. A good example is the occurrence of aflatoxin M1 (AFM1) in dairy products, which is due to the presence of aflatoxin B1 (AFB1) in the animal feed. Thus, milk-based foods, such as cheese and yogurts, may be contaminated with this toxin, which, although less toxic than AFB1, also exhibits hepatotoxic and carcinogenic effects and is relatively stable during pasteurization, storage and processing. For this reason, the establishment of allowed maximum limits in dairy products and the development of methodologies for its detection and quantification are of extreme importance. There are several methods for the detection of AFM1 in dairy products. Usually, the analytical procedures go through the following stages: sampling, extraction, clean-up, determination and quantification. For the extraction stage, the use of organic solvents (as acetonitrile and methanol) is still the most common, but recent advances include the use of the Quick, Easy, Cheap, Effective, Rugged, and Safe method (QuEChERS) and proteolytic enzymes, which have been demonstrated to be good alternatives. For the clean-up stage, the high selectivity of immunoaffinity columns is still a good option, but alternative and cheaper techniques are becoming more competitive. Regarding quantification of the toxin, screening strategies include the use of the enzyme-linked immunosorbent assay (ELISA) to select presumptive positive samples from a wider range of samples, and more reliable methods—high performance liquid chromatography with fluorescence detection or mass spectroscopy—for the separation, identification and quantification of the toxin.

Aflatoxin M1 detection by ELISA in raw and processed milk in Bangladesh

An analysis was accomplished to get information regarding presence of highly toxic and carcinogenic aflatoxin M1 (AFM1) in raw and processed samples of milk applying ELISA (enzyme linked immunosorbent assay). Investigation of a set of 100 samples (n=100) taken from different regional small-scale farms as well as grocery stores of Bangladesh containing three groups of milk including raw (n=50), pasteurized (n=25) and UHT (n=25), exhibited in total 53% AFM1 contamination where 70% contamination was found in raw milk ranging from 22.79−1489.28 ng/kg (mean value 699.07 ng/kg), 52% in pasteurized milk ranging from 18.11−672.18 ng/kg (mean value 99.77 ng/kg) and 20% in UHT milk ranging from 25.07−48.95 ng/kg (mean value 35.46 ng/kg). Among all the positive samples, 75% contaminated samples were above the European Communities prescribed limits (50 ng/kg) while having 25% samples still below this limit whereas 43% samples crossed the accepted limit of US regulations/Codex Alimentarius Commission regulations (500 ng/kg). Thus the findings of this study may lead to increase awareness regarding health impact of aflatoxin M1 and implementation of strict regulations by law enforcement bureaus of Bangladesh.