Signal-enhanced electrochemical sensor employing MWCNTs/CMK-3/AuNPs and Au@Pd core-shell structure for sensitive determination of AFB1 in complex matrix.

A sandwich electrochemical sensor was fabricated based on multi-walled carbon nanotubes/ordered mesoporous carbon/AuNP (MWCNTs/CMK-3/AuNP) nanocomposites and porous core-shell nanoparticles Au@PdNPs to achieve rapid and sensitive detection of AFB1 in complex matrices. MWCNTs/CMK-3/AuNP nanocomposite, which was prepared by self-assembly method, served as a substrate material to increase the aptamer loading and improve the conductivity and electrocatalytic activity of the electrode for the first signal amplification. Then, Au@PdNPs, which were synthesized by one-pot aqueous phase method, were applied as nanocarriers loaded with plenty of capture probe antibody (Ab) and signal molecule toluidine blue (Tb) to form the Au@PdNPs-Ab-Tb bioconjugates for secondary signal amplification. The sensing system could still significantly improve the signal output intensity even in the presence of ultra-low concentration target compound due to the dual signal amplification of MWCNTs/CMK-3/AuNP nanocomposites and Au@PdNPs-Ab-Tb. The method exhibited high selectivity, low detection limit (9.13 fg/mL), and strong stability to differentiate AFB1 from other mycotoxins. Furthermore, the sensor has been successfully applied to the quantitative determination of AFB1 in corn, malt, and six herbs, which has potential applications in food safety, quality control, and environmental monitoring.

Characterization of a Novel Monoclonal Antibody with High Affinity and Specificity against Aflatoxins: A Discovery from Rosetta Antibody-Ligand Computational Simulation.

Aflatoxin B1 (AFB1) accumulates in crops, where it poses a threat to human health. To detect AFB1, anti-AFB1 monoclonal antibodies have been developed and are widely used. While the sensitivity and specificity of these antibodies have been extensively studied, information regarding the atomic-level docking of AFB1 (and its derivatives) with these antibodies is limited. Such information is crucial for understanding the key interactions that are required for high affinity and specificity in aflatoxin binding. First, a 3D comparative model of anti-AFB1 antibody (Ab-4B5G6) was predicted from the sequence using RosettaAntibody. We then utilized RosettaLigand to dock AFB1 onto ten homology models, producing a total of 10,000 binding modes. Interestingly, the best-scoring mode predicted strong interactions involving four sites within the heavy chain: ALA33, ASN52, HIS95, and TRP99. Importantly, these strong binding interactions exclusively involve the variable domain of the heavy chain. The best-scoring mode with AFB1 was also obtained through AF multimer combined with RosettaLigand, and two interactions at TRP and HIS were consistent with those found by Rosetta antibody-ligand computational simulation. The role of tryptophan in π interactions in antibodies was confirmed through mutation experiments, and the resulting mutant (W99A) exhibited a >1000-fold reduction in binding affinity for AFB1 and analogs, indicating the effect of tryptophan on the stability of CDR-H3 region. Additionally, we evaluated the binding of two glycolic acid-derived molecular derivatives (with impaired hydrogen bonding potential), and these derivatives (AFB2-GA and AFG2-GA) demonstrated a very weak binding affinity for Ab-4B5G6. The heavy chain was successfully isolated, and its sensitivity and specificity were consistent with those of the intact antibody. The homology models of variable heavy (VH) single-domain antibodies were established by RosettaAntibody, and the docking analysis revealed the same residues, including Ala, His, and Trp. Compared to the potential binding mode of fragment variable (FV) region, the results from a model of VH indicated that there are seven models involved in hydrophobic interaction with TYR32, which is usually referred to as polar amino acid and has both hydrophobic and hydrophilic features depending on the circumstances. Our work encompasses the entire process of Rosetta antibody-ligand computational simulation, highlighting the significance of variable heavy domain structural design in enhancing molecular interactions.

A sandwich-type photoelectrochemical biosensor based on Ru(bpy)32+ sensitized In2S3 for aflatoxin B1 detection.

Aflatoxin B1 (AFB1), classified as a class I carcinogen, is a widespread mycotoxin that poses a serious threat to public health and economic development, and the food safety problems caused by AFB1 have aroused worldwide concern. The development of accurate and sensitive methods for the detection of AFB1 is significant for food safety monitoring. In this work, a sandwich-type photoelectrochemical (PEC) biosensor for AFB1 detection was constructed on the basis of an aptamer-antibody structure. A good photocurrent response was obtained due to the sensitization of In2S3 by Ru(bpy)32+. In addition, this sandwich-type sensor constructed by modification with the antibody, target detector, and aptamer layer by layer attenuated the migration hindering effect of photogenerated carriers caused by the double antibody structure. The aptamer and antibody synergistically recognized and captured the target analyte, resulting in more reliable PEC response signals. CdSe@CdS QDs-Apt were modified as a signal-off probe onto the sensor platform to quantitatively detect AFB1 with a "signal-off" response, which enhanced the sensitivity of the sensor. The PEC biosensor showed a linear response range from 10-12 to 10-6 g mL-1 with a detection limit of 0.023 pg mL-1, providing a feasible approach for the quantitative detection of AFB1 in food samples.

In silico studies and development of a protein-based electrochemical sensor for selective and sensitive detection of aflatoxin B1.

Proteins from different species have been docked with aflatoxin B1 (AFB1) and identified 3 proteins (prostaglandin-E(2)9-reductase from Oryctolagus uniculus, proto-oncogene serine/threonine-protein kinase Pim-1 and human immunoglobulin G (hIgG)) as potential candidates to develop an electrochemical sensor. Fluorescence spectroscopy experiments have confirmed the interaction of hIgG with AFB1 with an affinity constant of 4.6 × 105 M-1. As a proof-of-concept, hIgG was immobilized on carbon nanocomposite (carbon nanotube-nanofiber, CNT-F)-coated glassy carbon electrode (GCE). FT-IR spectra, HR-TEM and BCA assay have confirmed successful immobilization of hIgG on the electrode (hIgG@CNT-F/GCE). The preparation of this protein electrochemical sensor requires only 1 h 36 min, which is fast as compared with preparing an electro immunosensor. hIgG@CNT-F/GCE has displayed an excellent AFB1 limit of detection (0.1 ng/mL), commendable selectivity in the presence of two other mycotoxins (ochratoxin A and patulin) and the detection of  AFB1 in spiked peanuts and corn samples.

A disposable immunosensor array using cellulose paper assembled chemiresistive biosensor for simultaneous monitoring of mycotoxins AFB1 and FB1.

Due to the common contamination of multiple mycotoxins in food, which results in stronger toxicity, it is particularly important to simultaneously test for various mycotoxins for the protection of human health. In this study, a disposable immunosensor array with low-cost was designed and fabricated using cellulose paper, polydimethylsiloxane (PDMS), and semiconducting single-walled carbon nanotubes (s-SWCNTs), which was modified with specific antibodies for mycotoxins AFB1 and FB1 detection. The strategy for fabricating the immunosensor array with two individual channels involved a two-step protocol starting with the form of two kinds of carbon films by depositing single-wall carbon nanotubes (SWCNTs) and s-SWCNTs on the cellulose paper as the conductive wire and sensing element, followed by the assembly of chemiresistive biosensor with SWCNTs strip as the wire and s-SWCNTs as the sensing element. After immobilizing AFB1-bovine serum albumin (AFB1-BSA) and FB1-bovine serum albumin (FB1-BSA) separately on the different sensing regions, the formation of mycotoxin-BSA-antibody immunocomplexes transfers to electrochemical signal, which would change with the different concentrations of free mycotoxins. Under optimal conditions, the immunosensor array achieved a limit of detection (LOD) of 0.46 pg/mL for AFB1 and 0.34 pg/mL for FB1 within a wide dynamic range from 1 pg/mL to 20 ng/mL. Furthermore, the AFB1 and FB1 spiked in the ground corn and wheat extracts were detected with satisfactory recoveries, demonstrating the excellent practicality of this established method for simultaneous detection of mycotoxins.

A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products.

Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL-1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL-1 and 18 pg mL-1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.

Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification.

Nanobodies (Nbs) have shown great potential in immunodetection of small-molecule contaminants in food and environmental monitoring. However, the limited knowledge of the mechanism of Nbs binding to small molecules has hampered the development of high-affinity Nbs and assay improvement. We previously reported two homologous nanobodies Nb26 and Nb28 specific to aflatoxin B1 (AFB1), with the former exhibiting higher sensitivity in ELISA. Herein, Nb26 was selected as the model antibody to resolve its solution nuclear magnetic resonance (NMR) structure, and investigate its AFB1 recognition mechanism. The results revealed that Nb26 exhibits a typical immunoglobulin fold and its AFB1-binding interface is uniquely located in complementarity-determining region 3 (CDR3) and framework region 2 (FR2). This finding was applied to improve the binding activity of Nb28 against AFB1 by constructing two Nb28-based mutants A50V and S102D, resulting in 2.3- and 3.3-fold sensitivity enhancement over the wild type, respectively. This study develops an NMR-based strategy to analyze the underlying mechanism of Nb against AFB1, and successfully generated two site-modified Nbs with improved detection sensitivity. It is believed that this work could greatly expand the applications of Nbs by providing a way to enhance the binding activity.

Nanostructured graphitic carbon nitride based ultrasensing electrochemical biosensor for food toxin detection.

We report results of the studies related to the fabrication of thionine functionalized graphitic carbon nitride nanosheets based ultrasensing platform for food toxin (Aflatoxin B1, AfB1) detection. The synthesis of graphitic carbon nitride nanosheets (g-C3N4) was carried out by polycondensation of melamine followed by chemical exfoliation. Further, thionine was used for the functionalization of g-C3N4 (Thn/g-C3N4) and deposited electrophoretically onto the indium tin oxide (ITO) coated glass electrode. The fabricated Thn/g-C3N4/ITO electrode was covalently immobilized by EDC-NHS chemistry with anti-aflatoxin B1 (anti-AfB1) followed by blocking of non-specific sites using BSA molecules. For structural, morphological, functional and electrochemical properties analysis of synthesized nanomaterials and fabricated electrodes X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, atomic force microscopy and cyclic voltammetry techniques were used. The electrochemical response studies of the fabricated biosensing platform (BSA/anti-AfB1/Thn/g-C3N4/ITO) were carried out towards detection of AfB1 antigen using cyclic voltammetry technique. The obtained electrochemical results indicate that the fabricated biosensing electrode having ability to detect AfB1 with lower limit of detection of 0.328 fg mL-1, linear detection range in between 1 fg mL-1 to 1 ng mL-1, sensitivity of 4.85 µA log [ng-1 mL] cm-2 with stability upto 7 weeks.

Single-Line Flow Assay Platform Based on Orthogonal Emissive Upconversion Nanoparticles.

Lateral flow assay (LFA) has played pivotal roles in many emergency public safety incidents, such as coronavirus disease diagnostics; however, the present double-line (test and control line) design strategy for LFA strips greatly restricts their applications in high-throughput quantitative analysis because the limited sample diffusion distance on the strips constrains the number of test/control lines. Herein, a novel single-line-based LFA (sLFA) strip, which combines test and control line, is developed by exploiting an orthogonal emissive upconversion nanoparticle (UCNP) as a signal reporter on the test line, where one emission can be used as a reporting signal and the other as a calibrating signal. This UCNP-based test line with an interior reference also can play a validating role as a control line, and hence capturing antibodies are not needed for control lines, greatly saving fabrication costs. As a proof-of-concept, this novel sLFA strip is successfully explored to accurately and rapidly detect aflatoxin B1. Moreover, due to the removal of control lines, such a novel strategy greatly reduces the strip size, facilitating the design of a testing array for multiple detections of different samples. The test line herein is designed in a ring shape, and several test rings are assembled to be a chip for the testing of multiple samples. To our knowledge, this is the first demonstration of single-line-based LFA strips, which will significantly improve the detection capacities and accuracies and reduce the testing costs of LFA strips in real sample applications ranging from food analysis to in vitro diagnostics.

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

A homogeneous fluorescence quenching immunoassay is described for 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 10 min. Under optimized conditions, the visual and quantitative detection limits of the assay for AFM1 were 50 and 3.8 ng L-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.

Analysis of multiple mycotoxins-contaminated wheat by a smart analysis platform.

This study describes a smart analysis platform capable of quantitative measurements using a multiplex lateral flow strip. Using the multi-mycotoxin strip, five fungal toxins were simultaneously and quantitatively detected in naturally contaminated wheat. First, a matrix-based standard curve was established for the detection of aflatoxin B1 (AFB1), fumonisin B1 (FB1), T-2, deoxynivalenol (DON), and zearalenone (ZEN). Established on an open android system, the platform is able to read 6 lines on the strip simultaneously. The platform is equipped with a Quick Response code scanning model, which reads the established standard curves, and then rapidly quantify mycotoxins in naturally contaminated wheat. All the data and sample information are stored on a central server through the platform which is linked to the cloud. The limits of detection (LOD) for AFB1, FB1, T-2, DON, and ZEN in wheat were 4, 20, 10, 200, and 40 µg/kg and the visual cut off values was 20, 1000, 200, 4000, and 400 µg/kg, separately. To validate the platform and the multi-mycotoxin detection method, 10 wheat samples were analyzed and the results were in a good agreement with those obtained by LC-MS/MS. The platform will be a powerful tool for crop monitoring services.

Integrated graphene quantum dot decorated functionalized nanosheet biosensor for mycotoxin detection.

Decoration of graphene quantum dots (GQDs) on molybdenum disulfide (MoS2) nanosheets serves as an active electrode material which enhances the electrochemical performance of the analyte detection system. Herein, ionic surfactant cetyltrimethylammonium bromide (CTAB)-exfoliated MoS2 nanosheets decorated with GQD material are used to construct an electrochemical biosensor for aflatoxin B1 (AFB1) detection. An antibody of AFB1 (aAFB1) was immobilized on the electrophoretically deposited MoS2@GQDs film on the indium tin oxide (ITO)-coated glass surface using a crosslinker for the fabrication of the biosensor. The immunosensing study investigated by the electrochemical method revealed a signal response in the range of 0.1 to 3.0 ng/mL AFB1 concentration with a detection limit of 0.09 ng/mL. Also, electrochemical parameters such as diffusion coefficient and heterogeneous electron transfer (HET) were calculated and found to be 1.67 × 10-5 cm2/s and 2 × 10-5 cm/s, respectively. The effective conjugation of MoS2@GQDs that provides abundant exposed edge sites, large surface area, improved electrical conductivity, and electrocatalytic activity has led to an excellent biosensing performance with enhanced electrochemical parameters. Validation of the fabricated immunosensor was performed in a spiked maize sample, and a good percentage of recoveries within an acceptable range were obtained (80.2 to 98.3%).Graphical abstract.

Sensitive electrochemical detection of aflatoxin B1 using DNA tetrahedron-nanostructure as substrate of antibody ordered assembly and template of aniline polymerization.

A novel strategy for AFB1 detection in grains was proposed based on DNA tetrahedron-structured probe (DTP) and horseradish peroxidase (HRP) triggered polyaniline (PANI) deposition. Briefly, the DNA tetrahedron nanostructures were assembled on the gold electrode, with carboxylic group designed on top vertex of them. The carboxylic group was conjugated with the AFB1 monoclonal antibody (mAb) to form DTP. The test sample and a known fixed concentration of HRP-labeled AFB1 were mixed and they compete for binding to DTP. The HRP assembled on the gold electrode catalyzed the polymerization of aniline on DTP. AFB1 in grains could be determined by using PANI as electrochemical signal molecules. Interestingly, DNA tetrahedron-structure, which has mechanical rigidity and structural stability, can improve antigen-antibody specific recognition and binding efficiency through the use of mAb ordered assembly. Meanwhile, nucleic acid backbone with a large amount of negative charge is good template for aniline polymerization under mild conditions.

Nanobody-based electrochemical competitive immunosensor for the detection of AFB1 through AFB1-HCR as signal amplifier.

A novel nanobody (Nb)-based voltammetric immunosensor coupled with horseradish peroxidase concatemer-modified hybridization chain reaction (HRP-HCR) signal amplifying system is described to realize the rapid and ultrasensitive detection of AFB1. To design such an immunoassay, anti-AFB1 Nbs with smaller molecular size were coated densely onto the surface of Au nanoparticle-tungsten disulfide-multi-walled carbon nanotubes (AuNPs/WS2/MWCNTs) functional nanocomposites as an effective molecular recognition element, whereas AFB1-streptavidin (AFB1-SA) conjugates were ingeniously bound with biotinylated HCR dsDNA nanostructures as the competitor, amplifier, and signal report element. In the presence of AFB1 targets, a competitive immunoreaction was performed between the analyte and AFB1-SA-labeled HCR (AFB1-HCR) platform. Upon the addition of SA-modified polyHRP (SA-polyHRP), AFB1-HCR nanostructures containing abundant biotins were allowed to cross-link to a quantity of HRP by streptavidin-biotin chemistry for signal amplification and signal conversion. Under optimal conditions, the immunosensor displayed a good linear correlation toward AFB1 ranging from 0.5 to 10 ng mL-1 with a sensitivity of 2.7 µA • (mL ng-1) and an ultralow limit of detection (LOD) of 68 fg mL-1. The specificity test showed that the AFB1 immunosensor had no obvious cross-reaction with OTA, DON, ZEN, and FB1. The signal of this sensor decreased by 10.18% in 4 weeks indicating satisfactory stability, and its intra- and inter-laboratory reproducibility was 3.42~10.35% and 4.03%~12.11%, respectively. This biosensing system will open up new opportunities for the detection of AFB1 in food safety and environmental analysis and extend a wide range of applications in the analysis of other small molecules. Graphical abstract.

Sensitive Aflatoxin B1 Detection Using Nanoparticle-Based Competitive Magnetic Immunodetection.

Food and crop contaminations with mycotoxins are a severe health risk for consumers and cause high economic losses worldwide. Currently, different chromatographic- and immuno-based methods are used to detect mycotoxins within different sample matrices. There is a need for novel, highly sensitive detection technologies that avoid time-consuming procedures and expensive laboratory equipment but still provide sufficient sensitivity to achieve the mandated detection limit for mycotoxin content. Here we describe a novel, highly sensitive, and portable aflatoxin B1 detection approach using competitive magnetic immunodetection (cMID). As a reference method, a competitive ELISA optimized by checkerboard titration was established. For the novel cMID procedure, immunofiltration columns, coated with aflatoxin B1-BSA conjugate were used for competitive enrichment of biotinylated aflatoxin B1-specific antibodies. Subsequently, magnetic particles functionalized with streptavidin can be applied to magnetically label retained antibodies. By means of frequency mixing technology, particles were detected and quantified corresponding to the aflatoxin content in the sample. After the optimization of assay conditions, we successfully demonstrated the new competitive magnetic detection approach with a comparable detection limit of 1.1 ng aflatoxin B1 per ml sample to the cELISA reference method. Our results indicate that the cMID is a promising method reducing the risks of processing contaminated commodities.

Development of a ZnCdS@ZnS quantum dots-based label-free electrochemiluminescence immunosensor for sensitive determination of aflatoxin B1 in lotus seed.

In this study, we designed a ZnCdS@ZnS quantum dots (QDs)-based label-free electrochemiluminescence (ECL) immunosensor for sensitive determination of aflatoxin B1 (AFB1). A Nafion solution assembled abundant QDs on the surface of a Au electrode as ECL signal probes, with specially coupled anti-AFB1 antibodies as the capturing element. As the reduction reaction between S2O82- in the electrolyte and QDs on the electrode led to ECL emission, the decreased ECL signals resulting from target AFB1 in the samples were recorded for quantification. We evaluated electrochemical impedance spectroscopy and ECL measurements along each step in the construction of the proposed immunosensor. After systematic optimization of crucial parameters, the ECL immunosensor exhibited a good sensitivity, with a low detection limit of 0.01 ng/mL for AFB1 in a wide concentration range of 0.05-100 ng/mL. Testing with lotus seed samples confirmed the satisfactory selectivity, stability, and reproducibility of the developed ECL immunosensor for rapid, efficient, and sensitive detection of AFB1 at trace levels in complex matrices. This study provides a powerful and universal analytical platform for a variety of analytes that can be used in broad applications for real-time analysis, such as food and traditional Chinese medicine safety testing, environmental pollution monitoring, and disease diagnostics. Graphical abstract Development of a ZnCdS@ZnS quantum dots based label-free electrochemiluminescence immunosensor for sensitive detection of aflatoxin B1 in lotus seed.

Fluorometric lateral flow immunoassay for simultaneous determination of three mycotoxins (aflatoxin B1, zearalenone and deoxynivalenol) using quantum dot microbeads.

A fluorometric lateral flow immunoassay (LFA) is described for the simultaneous determination of the mycotoxins aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON). The method is based on the use of CdSe/SiO2 quantum dot microbeads (QBs) with a mean diameter of 106 nm. These have strong red luminescence (with excitation/emission peaks at 365/622 nm) which results in enhanced sensitivity. The QBs binding with monoclonal antibodies (mAbs) as the signal probes can react specifically with AFB1, ZEN and DON, respectively. There is an inverse correlation between the fluorescence signal intensity of test line and the analyte content, which can realize the quantitative analysis of analytes within 15 min. The limits of detection in solution are 10, 80 and 500 pg mL-1 for AFB1, ZEN and DON, respectively. Besides, the average recoveries from spiked feed range from 85.5 to 119.0%, and the relative standard deviations are less than 16.4% for both intra- and inter-day assays. The method was used to analyze naturally contaminated feedstuff, and this resulted in a good agreement with data obtained by LC-MS/MS. Graphical abstractSchematic representation of a fluorometric method for the simultaneous determination of three mycotoxins. Quantum dot microbeads (QBs) binding with monoclonal antibodies (mAbs) are signal probes. There is an inverse correlation between the fluorescence intensity of test line and the analyte concentration.

p-Bromophenol-Enhanced Bienzymatic Chemiluminescence Competitive Immunoassay for Ultrasensitive Determination of Aflatoxin B1.

Aflatoxin B1 (AFB1) contamination is one of the most critical global issues in food safety. The high carcinogenic nature necessitates rapid and specific methods for the determination of AFB1 in foodstuffs at ultratrace levels. Here, we report an enhanced bienzymatic chemiluminescence competitive immunoassay for ultrasensitive and high-throughput determination of AFB1. In this assay, protein G was first coated on the wells of a microplate for recognizing the Fc fragment of anti-AFB1 mAbs to reduce the antibody dosage and guarantee high immunological reaction efficiency. The target AFB1 competed with glucose oxidase labeled AFB1 for the limited anti-AFB1 mAbs in the wells of the microplate. p-Bromophenol was employed as an enhancer to obtain intense and long-lasting chemiluminescence. The utilization of an enhancer and bienzymatic catalysts effectively improved the detection sensitivity. The developed method offered a good linearity over 5 orders of magnitude, a detection limit of 5 pg L-1, and a relative standard deviation of 1.9% for AFB1. The application of the developed method to the analysis of grain samples gave quantitative recoveries from 94.0% to 97.0%. The developed method provides a universal platform for high-throughput, ultrasensitive, and high specific detection of pollutants or nutrients in foods.

Electrochemical Aflatoxin B1 immunosensor based on the use of graphene quantum dots and gold nanoparticles.

Electrochemical immunosensor for aflatoxin B1 (AFB1) is described that uses a composite prepared from graphene quantum dots (GQDs) and gold nanoparticles (Au NPs). The GQD-AuNP conjugate was obtained by using 2-aminothiophenol (ATP) as a linker where the carboxy groups of GQD bind to the amino groups of crosslinker via conjugation of thiol binding to the AuNP. To evaluate the conjugation of the GQD-AuNP composite, Fourier transform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM) was applied. The composite was placed on an indium tin oxide (ITO) electrode and then modified with an antibody against AFB1. By using hexacyanoferrate as the electrochemical probe, the sensor works in the 0.1 to 3.0 ng mL-1 AFB1 concentration range, is highly specific, has good reproducibility and acceptable stability. The biosensor was applied to the analysis of (spiked) maize samples. Conceivably, the method can be utilized to sense other mycotoxins by using their respective antibodies. Graphical abstract Schematic presentation of electrochemical immunosensor for Aflatoxin B1 (AFB1) detection developed by antibodies of AFB1 (anti-AFB1) immobilization on graphene quantum dots (GQDs)-gold nanoparticles (AuNPs) composite deposited by electrophoretic deposition technique on an Indium tin oxide (ITO) surface.

Simultaneous detection of aflatoxin B1, ochratoxin A, zearalenone and deoxynivalenol in corn and wheat using surface plasmon resonance.

Mycotoxins are toxic metabolites produced by fungi or molds, which may cause serious harm to human health through polluted cereal foods. In order to measure the typical mycotoxin contaminations in wheat and corn, a surface plasmon resonance (SPR) method was established using SPR sensor chip that was fabricated based on self-assembled monolayer. The minimum detection limit of aflatoxin B1, ochratoxin A, zearalenone and deoxynivalenol were identified as 0.59 ng/mL, 1.27 ng/mL, 7.07 ng/mL and 3.26 ng/mL, respectively. The cross-reactivity for all four mycotoxins were demonstrated to be low. Moreover, the test data were compared with HPLC-MS/MS confirmatory analysis results and good agreement was found between them. In conclusion, the SPR method for simultaneously detecting four mycotoxins has been developed with high sensitivity, good linearity and specificity, which can meet the detection requirements of cereal foods.