Anti-aflatoxin B1 Research Articles

Development of a chimeric aptamer and an AuNPs aptasensor for highly sensitive and specific identification of Aflatoxin B1

In this study, a bivalent anti-Aflatoxin B1 (AFB1) chimeric aptamer (B72) was firstly engineered by a rational truncation and splicing-based post-selection strategy. Compared with that of the originally-selected anti-AFB1 aptamer (B50), a 188-fold enhanced affinity of B72 was revealed by microscale thermophoresis (MST) assay. In addition, isothermal titration calorimetry (ITC) research and molecular docking simulation affirmed consistently that B72 possessed dual binding sites for AFB1. A gold nanoparticles (AuNPs) aptasensor was developed to further validate the real-world applicability of the chimeric aptamer by colorimetric assay of AFB1 based on the peroxidase-like activity of AuNPs to catalyze 3,3,5,5-tetramethylbenzidine (TMB) with the assistance of H2O2. The aptasensor could identify AFB1 in corn oil with a limit of detection (LOD) of 1.88 nM and a linear range of 5–5120 nM. Moreover, good recovery rates of 91.5–117.6% and low relative standard deviations below 9% were obtained, suggesting the good accuracy of the developed aptasensor. The study could thus be referred as a universal post-selection strategy for engineering high affinity aptamers and constructing novel aptamer-based biosensing platforms for highly sensitive and specific analysis of other targets.

Unravelling the antifungal and anti-aflatoxin B1 mechanism of chitosan nanocomposite incorporated with Foeniculum vulgare essential oil

The study reports the antifungal and aflatoxin B1 inhibitory efficacy of chemically characterized chitosan-based nanoencapsulated Foeniculum vulgare Mill. essential oil (Ne-FvEO), and its effect on sensory and nutritional properties of Sorghum bicolor. The Ne-FvEO was characterized through SEM, FTIR and XRD. The Ne-FvEO demonstrated superior antifungal (0.4 μl/ml) and aflatoxin B1 inhibitory (0.3 μl/ml) performance than the free FvEO. The biochemical studies reveal the significant alteration in ergosterol content, ions leakage, mitochondrial membrane potential, C-sources utilization and antioxidant defense system in A. flavus exposed to Ne-FvEO. The 3D modeling of the Nor-1 gene product was done using the I-TASSER server and validated by the Ramachandran plot. The in-situ result reveals that the Ne-FvEO significantly preserved the nutritional and sensory characteristics of S. bicolor seeds. Therefore, Ne-FvEO could be used as a green preservative agent to enhance the shelf-life of the food commodities.

Quartz Crystal Microbalance Detection of Aflatoxin B1 by Self-Assembled Monolayer Technique

Background: New biosensor techniques allowing detection of low concentrated substances show a great variety nowadays. The construction of a system with modified quartz as a part of a Quartz Crystal Microbalance (QCM) techniques helps the detection and confirmation of low toxin concentrations in a sample. Objective: The study aims to allow the application of methods for preparation and modification of the gold surface of piezoelectric crystal for detection of aflatoxin B1(AFB1) in the concentration range 0.2 - 2.0 µg/L by QCM technique (quartz - crystal microbalance). Methods: The procedure for the preparation of quartz crystal sensors for experimental purposes was performed. The quartz surface was activated and covered with self-assembled monolayer to immobilize antibody (rabbit anti-aflatoxin B1) for the detection of antigen - antibody reaction. Results: The G” corresponds to viscous properties of the material, during applied deformation of the material in the presence of different concentrations, which revealed in the sensitivity of the used resonator. Conclusion: Detection of toxic pollutants may be achieved via QCM methods, ultrasound resonator and piezoelectric quartz techniques for measurement. These techniques allow detection of significantly low concentrations of toxic pollutants, in particular, AFB1, compared to analysis with direct and indirect ELISA immunoassays.

Phytochemical analysis of Linaria purpurea (L.) Mill. and inhibitory activity on the production of aflatoxin B1 (AFB1) in Aspergillus flavus Link. of one of its metabolites, antirrhinoside

In this work, the first phytochemical study on the total polar fraction of Linaria purpurea (L.) Mill. was performed by means of column chromatography and NMR and MS analysis. Seven compounds were identified i.e. pheophytin a (1), methyl-pheophorbide a (2), linaride (3), antirrhide (4), antirrhinoside (5), linarioside (6) and shikimic acid (7), belonging to three different classes of natural compounds. Compound (2) represents a new compound for the family as well as compound (1) is for the genus and compounds (3, 6, 7) are for the species. The chemosystematic and ethnopharmacological relevance of these results were widely discussed. In addition, compound (5) was tested, for the first time, for its potential antifungal activity, showing to drastically reduce the production of aflatoxin B1 in Aspergillus flavus Link. This further property of compound (5) makes it a potential natural and green anti-aflatoxin B1 agent to be used in the food industry.

Nanoencapsulated plant-based bioactive formulation against food-borne molds and aflatoxin B1 contamination: Preparation, characterization and stability evaluation in the food system

A novel synergistic formulation (TML) based on the combination of thymol (T), methyl cinnamate (M), and linalool (L) has been prepared using the mixture design assay. Nanoencapsulation of developed formulation TML (Ne-TML) was prepared and characterised by SEM, XRD and FTIR. The Ne-TML was assessed for its antifungal and anti-aflatoxin B1 potential in vitro and in the food systems (Pennisetum glaucum L.), and also examined its effects on organoleptic properties. The Ne-TML cause complete inhibition of growth and AFB1 production at 0.3 μl/ml and 0.2 μl/ml. In-situ results revealed that Ne-TML exhibited maximum protection from fungal (75.40%) and aflatoxin B1 contamination (100%) at 0.3 μl/ml during six months of storage. The speculated antifungal mode of action of Ne-TML was related to the decrease in ergosterol content, membrane ions leakage, impairment in carbon-source utilization, mitochondrial functioning, anti-oxidative defence system (SOD, CAT, and GR) and Ver-1 gene of aflatoxin B1 biosynthesis.

Screening of Anti-Aflatoxin B1 ScFv Based on Phage Display Technology and Analysis of Its Protein Structure

Screening of Anti-Aflatoxin B1 ScFv Based on Phage Display Technology and Analysis of Its Protein Structure

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody-Ligand Interaction Using Chain Shuffling.

A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.

Development of Hybrid IgG-Aptamer Sandwich Immunoassay Platform for Aflatoxin B1 Detection and Its Evaluation Onto Various Field Samples.

The present study was aimed to develop a novel antibody-aptamer based hybrid detection strategy for specific and sensitive detection of aflatoxin B1 (AFB1) from contaminated food grains. The study comprises generation of ssDNA aptamers and anti-AFB1 IgG against AFB1 toxin. The generated bio-probes (aptamers and antibodies) were further characterized for their specificity and sensitivity using indirect ELISA. The generated aptamers namely AFB1a and AFB1b showed prominent reactivity and selectivity against AFB1 toxin. These aptamers were further characterized for their secondary structures and dG values were determined as −4.6 and −2.75 Kcal/mol, respectively. The detection limit (LOD) of AFB1a and anti-AFB1 IgG was determined as 5 and 10 ng/mL, respectively. The characterized aptamers and antibodies against AFB1 were used to develop the sandwich immunoassay. Anti AFB1 IgG was used as a capturing antibody whereas anti-AFB1a aptamer was used as its revealing partner in the assay. The limit of detection (LOD) of the immunoassay was determined to be 5 ng/mL of AFB1 standard toxin and showed no cross-reactivity with closely related mycotoxins. To assess the reliability of the developed method, several field samples contaminated with aflatoxin B1 was included in the study and results were validated with commercial AFB1-ELISA Kit. Additionally, the spiking studies were also carried out to demonstrate the consistency and dependability of the developed hybrid sandwich immunoassay wherein the toxins recovered were found to be ranging between 73 and 98.80% with the LOD at 5 ng/mL. In conclusion, the developed method may find the better utility in routine food testing laboratories for assessment of AFB1.

In Vivo Assessment of Gamma Rays, Electron-beam Irradiation plus a Commercial Toxin Binder (Milbond-TX) As an Anti-Aflatoxin B1 in a Chicken Model

In Vivo Assessment of Gamma Rays, Electron-beam Irradiation plus a Commercial Toxin Binder (Milbond-TX) As an Anti-Aflatoxin B1 in a Chicken Model

A highly sensitive amperometric immunosensor probe based on gold nanoparticle functionalized poly (3, 4-ethylenedioxythiophene) doped with graphene oxide for efficient detection of aflatoxin B1

In the present work, a platform of Poly (3, 4-ethylenedioxythiophene) (PEDOT) and graphene oxide (GO) composite decorated with spherical gold nanoparticles (AuNPs) has been developed for rapid electrochemical detection of aflatoxin B1 (AFB1). Electrochemical deposition of EDOT onto glassy carbon electrode (GCE) keeping GO as dopant followed by introduction of AuNPs has been achieved resulting in nanohybrid AuNPs/PEDOT-GO electrode. The antibody anti-aflatoxin B1 (anti-AFB1) has been further covalently immobilized onto the surface of AuNPs/PEDOT-GO using EDC/NHS coupling. The morphological and surface characteristics have been studied using Field Emission Scanning Microscopy (FESEM) and contact angle measurements. Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) studies have been carried out to investigate the electro-catalytic behavior of the modified electrodes. The heterogeneous rate constant (ks) and transfer coefficient (α) have been determined by using Laviron’s method. The proposed immunosensor exhibits a very high sensitivity of 0.989 μA ng mL−1 and 0.397 μA ng mL−1 within two linear range of 0.5–20 ng mL−1 and 20–60 ng mL−1, respectively.

A Low-Cost Label-Free AFB1 Impedimetric Immunosensor Based on Functionalized CD-Trodes

This work describes the investigation of a label-free immunosensor for the detection of aflatoxin B1 (AFB1). CD-trodes (electrodes obtained from recordable compact disks) were used as low-cost and disposable transducers after modification with a self-assembled monolayer (SAM) of lipoic acid. The anti-aflatoxin B1 antibody was immobilized via EDC/NHS activation, followed by blocking with bovine serum albumin and immunoassays with AFB1. The optimization of analytical parameters and the detection were carried out using electrochemical impedance measurements. Using chemometric tools, the best conditions for the immunosensor development were defined as: anti-AFB1 antibody at 1:2000 dilution and surface blocking with 0.5% bovine serum albumin, both incubated for 1 h, and antibody–antigen immunoreaction for 30 min. The impedimetric immunosensor showed a linear range from 5 × 10−9 to 1 × 10−7 mol·L−1 (1.56–31.2 ng·mL−1), limit of detection and limit of quantification, respectively, 3.6 × 10−10 and 1.1 × 10−9mol·L−1 (0.11 and 0.34 ng·mL−1). The proposed immunosensor was applied to analyze peanut samples.

Anti-aflatoxin B1 effects of Shirazi thyme (Zataria multiflora) in broilers: evaluation of performance and liver histopathology

An experiment was conducted to study the effect of <em>Zataria multifora</em> (ZM) on the performance and liver histopathology of broiler chickens contaminated with aflatoxin B1 (AFB1). One hundred and sixty Ross 308 male broilers (one-day-old) were divided into four treatment groups with four replicates with 10 birds in each replicate. The chickens were reared on the floor for 35 days. The groups were contaminated with AFB1 at two different concentrations,<em> i.e.,</em> 0 and 1000 ppb, and fed ZM in their feed at the concentrations of 0 and 20 gr Kg 1. The evaluated performance parameters were subjected to a completely randomized design with a 2×2 factorial arrangement of the treatments using SAS software (version 9/1). AFB1 had a statistical lowering effects on the feed intake, body weight, body weight gain and average weight of the carcass, thigh, chest, bursa of fabricius, back and neck. Also, the weights of liver, gizzard, pancreas, proventriculus, abdominal fat, full intestine, and heart were increased with AFB1 (P<0.05). In histopathological evaluations, the liver of chickens that received feed containing AFB1 showed multifocal and varied cytoplasmic vacuolization, severe fatty change, degenerating foci, fibrosis of the portal regions, and bile duct hyperplasia. The variables that were evaluated in this study showed that ZM had significant efficacy in diminishing the aflatoxins negative effects on the chickens.

Sensitive QD@SiO2-based immunoassay for triplex determination of cereal-borne mycotoxins

A sensitive tool for simultaneous quantitative determination of three analytes in one single well of a microtiter plate is shown for the first time. The developed technique is based on use of colloidal quantum dot enrobed into a silica shell (QD@SiO2) derivatives as a highly responsive label. Silica-coated quantum dots were prepared and subsequently modified via the co-hydrolysis with tetraethylorthosilicate (TEOS) and various organosilane reagents. Different surface modification schemes were compared in terms of applicability of the obtained particles for the multiplex immunoassay, e.g. stability and simplicity of their conjugation with biomolecules. As model system a multiplex immunosorbent assay for screening of three mycotoxins (deoxynivalenol, zearalenone and aflatoxin B1) in cereal-based products was realized via a co-immobilization of three different specific antibodies (anti- deoxynivalenol, anti-zearalenone and anti-aflatoxin B1) in one single well of a microtiter plate. Mycotoxins were simultaneously determined by labelling their conjugates with QD@SiO2 emitting in different parts of the visible spectrum. The limits of detection for the simultaneous determination were 6.1 and 5.3, 5.4 and 4.1, and 2.6 and 1.9µgkg−1 for deoxynivalenol, zearalenone and aflatoxin B1 in maize and wheat, respectively. As confirmatory method, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used.

Screening of self-assembled monolayer for aflatoxin B1 detection using immune-capacitive sensor

A capacitive biosensor was used for detection of aflatoxin B1. Two different methods for cleaning gold electrodes were evaluated using cyclic voltammetry in the presence of ferricyanide as redox couple. The methods involve use of a sequence of cleaning steps avoiding the use of Piranha solution and plasma cleaner. Anti-aflatoxin B1 was immobilized on self-assembled monolayers (SAM). The immune-capacitive biosensor is able to detect aflatoxin B1 concentrations in a linear range of 3.2×10−12M to 3.2×10−9M when thiourea was used to form the SAM; 3.2×10−9M to 3.2×10−7M when thioctic acid was used. When the gold surface was isolated with tyramine-electropolymerization linear ranges of 3.2×10−13M to 3.2×10−7M and 3.2×10−9M to 3.2×10−7M where obtained, respectively. The results obtained show the difference in linear range, limit of detection, and limit of quantification when different self-assembled monolayers are used for aflatoxin B1 detection.

Electrochemical piezoelectric reusable immunosensor for aflatoxin B1 detection

A competitive, reusable, reliable and sensitive immunosensor based on electrochemical quartz crystal microbalance (EQCM) was developed for the detection of aflatoxin B1 (AFB1). The sensing platform was developed using a primary monoclonal anti-aflatoxin antibody (anti AFB1) covalently immobilized on a monolayer of 4-amino thiophenol self assembled on a gold coated quartz crystal electrode (anti AFB1/4-ATP/Au). The reusability was achieved by a sandwiched system using secondary rabbit-immunoglobulin antibodies (r-IgGs) tagged with core shell of gold coated iron oxide nanoparticles (r-IgG–Au-Fe3O4), which can be used to regenerate the bioelectrode. A competitive mode was employed, between free and coated AFB1 for fixed concentration of nanoparticle conjugate (r-IgG–Au–Fe3O4). After the competitive interaction, immunoelectrode was washed with phosphate saline buffer (PBS) at pH 7.4 and examined by electrochemical quartz crystal microbalance-cyclic voltammetry (EQCM-CV). Under the optimized conditions, the fabricated immunosensor can be used to detect AFB1 quantitatively with a linear range of 0.05 to 5ngmL−1. For reliability, the fabricated immuno sensor was tested using cereal samples spiked with different concentrations of AFB1. In addition, this immuno electrode displays good reproducibility, and storage stability. The above immunosensor was regenerated with negligible loss in activity through removal of the r-IgG–Au–Fe3O4 conjugate using a strong magnet. It is shown that theAFB1/BSA/aAFB1/4-ATP/Au immuno sensor is a new approach for developing sensitive reliable and reusable electrochemical piezoelectric immunosensors.

Highly Sensitive FRET-Based Fluorescence Immunoassay for Detecting of Aflatoxin B1 Using Magnetic/Silica Core-Shell as a Signal Intensifier.

Recently, some new nanobiosensors using different nanoparticles or microarray systems for detection of mycotoxins have been designed . However, rapid, sensitive and early detection of aflatoxicosis would be very helpful to distinguish high-risk persons. We report a highly sensitive competitive immunoassay using magnetic/silica core shell as a signal intensifier for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from Cd/Te quantum dots (antiaflatoxin B1 antibody immobilized on the surface of Cd/Te quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The specific immune-reaction between the anti-aflatoxin B1 antibody on the QDs and the labeledaflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photo-excitation of the QDs. Using magnetic/silica core shell to intensify the obtained signal is the novelty of this study. Cd/Te QDs were synthesized by the simultaneous reduction of cadmium chloride and tellurium in the presence of sodium borohydride under nitrogen atmosphere. Magnetic nanoparticles were synthesized using FeSO4 and FeCl3 (1:2 molar ratio) and ammonia as an oxidizing agent under nitrogen atmosphere. The prepared magnetic nanoparticles shelled by silica using tetraethoxysilane in the presence of ammonia. Nanoparticles synthesis and monodispersity confirmed by TEM. Immobilization of Cd/Te QDs to antibodies and labeling of aflatoxin B1-albumin by Rho 123 were performed by EDC/NHS reaction in reaction mixture buffer, pH 6, at room temperature. By using the magnetic/silica core shell sensitivity of the system changed from 2×10-11 in our previous study to 2×10-12 in this work. The feasibility of the method established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the decreased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spiked samples, over the range of 0.01-0.06 μmol.mL-1. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require multiple separation steps and excessive washing.

Effect of Carum copticum essential oil on growth and aflatoxin formation by Aspergillus strains

The objectives of this study were to determine the antiaflatoxin B1 activity in vitro of the essential oil (EO) extracted from the seeds of Carum copticum and to evaluate its antifungal activity in vivo as a potential food preservative. The C. copticum EO exhibited noticeable inhibition on dry mycelium and synthesis of aflatoxin B1 (AFB1) by Aspergillus flavus, completely inhibiting AFB1 production at 4 μL/mL. C. copticum EOs showed the lowest percentages of decayed cherry tomatoes for all fungi compared with the control at 100 μL/mL with values of 5.01 ± 67% for A. flavus and 5.98 ± 54% for Aspergillus niger. The results indicated that the percentage of infected fruits is significantly (p < 0.01) reduced by the EO at 16°C for 30 days. In this case, the oil at 100 μL/mL concentration showed the highest inhibition of fungal infection with a value of 80.45% compared with the control. Thus, the EO of dill could be used to control food spoilage and as a potential source of food preservative.

Rapid detection method for aflatoxin B1 in soybean sauce based on fluorescent microspheres probe

Soybean sauce, a Chinese traditional and daily condiment, is often contaminated by aflatoxin B1. An extract-free immunochromatographic assay was proposed based on fluorescent microspheres probe for the' detection of aflatoxin B1 in soybean sauce. The probe was prepared by coupling fluorescent microspheres with anti-aflatoxin B1 antibody by the 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride-mediated method. The background from the soybean sauce sample on strip was eliminated because of the optical property of the probe. The sample without extracting procedure was directly detected by diluting with 10% methanol solution. The visible detection limit for the qualitative analysis of aflatoxin B1 in the proposed method was 2.5 μg/L, which was lower than the maximum level of 5 μg/L set by the Chinese government. The results were well agreed with those obtained by liquid chromatography-mass spectrometry (LC-MS). The method showed satisfactory characteristics, such as rapid detection, easy operation, and high sensitivity, and can thus be applied for the large-scale and on-site screening of soybean sauce contaminated with aflatoxin B1. To our knowledge, this report is the first one on the qualitative detection of aflatoxin B1 in dark colored food samples directly by fluorescent microspheres probe-based immunochromatography.

Amperometric Immuno-sensor For Detection Of Toxin Aflatoxin B1 Based On Polyaniline Probe Modified With Mc-IgGs-a-AFB1 Antibodies

An amperometric sensor for detection of toxin aflatoxin B1 from aspergillus flavus based on conducting polyaniline probe using monoclonal anti-aflatoxin B1 (Mc-IgGs-a-AFB1) antibodies after activation with 3% Bovine Serum Albumin (BSA) through electrochemical polymerization has been proposed. The electrode was fabricated by immobilizing Mc-IgGs-a-AFB1 antibodies molecules onto electrode surface and characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM) and fourier transform infrared spectroscopic (FT-IR) etc. The proposed amperometric immune-sensor has demonstrated excellent electro-analytical properties relative to Aflatoxin B1 in a linear range from 0.20 to 1.30 AngmL -1 with a relatively low detection limit of 0.059 AngmL -1 . The present study will help in improving for quantitative determination of mycotoxins in food samples may provide significant improvements in quality control of food safety through a simple, rapid, and sensitive testing system for agricultural products monitoring.

Vaccination of heifers with anaflatoxin improves the reduction of aflatoxin b1 carry over in milk of lactating dairy cows.

It was previously reported that injection of anaflatoxin B1 (AnAFB1) conjugated to keyhole limpet hemocyanin (KLH), together with Freund's adjuvant, was effective in inducing in cows a long lasting titer of anti-aflatoxin B1 (AFB1) antibodies (Abs), cross-reacting with other aflatoxins, which were able to hinder, proportionally to their titer, the secretion of aflatoxin M1 (AFM1) into the milk of cows continuously fed with AFB1. According to anti-AFB1 Ab titer, 50% of the vaccinated cows were recognized as high responder animals. In an attempt to prepare a more effective formulation for vaccination of cows, it was compared the immunogenicity, in Holstein Friesian heifers, of AnAFB1 covalently conjugated to KLH or to recombinant diphtheria toxin (CRM197) molecules, and injected together with various adjuvants. This study demonstrated that injection of AnAFB1 conjugated to KLH and mixed with complete (priming) and incomplete Freund's adjuvant (boosters), as in the previous schedule of immunization, was the most effective regimen for inducing Ab responses against AFB1, although pre-calving administration could increase the effectiveness of vaccination, resulting in 100% high responder animals. After one booster dose at the beginning of the milk production cycle, anti-AFB1 Ab titers were comparable to those recorded at the end of the immunization schedule, and proved to be effective in reducing significantly AFB1 carry over, as AFM1, from feed to milk. Pre-calving vaccination of dairy heifers with conjugated AnAFB1, adjuvated with complete and incomplete Freund's adjuvant, may represent the most effective tool for preventing the public health hazard constituted by milk and cheese contaminated with aflatoxins.