Rapid Detection Of Aflatoxin B1 Research Articles

DNA-functionalized cryogel based colorimetric biosensor for sensitive on-site detection of aflatoxin B1 in food samples

Hydrogel biosensors present numerous advantages in food safety analysis owing to their remarkable biocompatibility, cargo-loading capabilities and optical properties. However, the current drawbacks (slow target responsiveness and poor mechanical strength) restricted their further utilization at on-site detection of targets. To address these challenges, a DNA-functionalized cryogel with hierarchical pore structures is constructed to improve the reaction rate and the robustness of hydrogel biosensor. During cryogel preparation, ice crystals serve as templates, shaping interconnected hierarchical microporous structures to enhance mass transfer for faster responses. Meanwhile, in the non-freezing zone, concentrated monomers create a dense cross-linked network, strengthening cryogel matrix strength. Accordingly, a colorimetric biosensor based on DNA cryogel has been developed as a proof of concept for rapid detection of aflatoxin B1 (AFB1) in food samples, and an excellent analytical performance was obtained under the optimized conditions with a low detection limit (1 nM), broad detection range (5–100 nM), satisfactory accuracy and precision (recoveries, 81.2–112.6 %; CV, 2.75–5.53 %). Furthermore, by integrating with a smartphone sensing platform, a portable device was created for rapid on-site measurement of target within 45 min, which provided some insight for hydrogel biosensors design.

Smartphone-integrated ratiometric sensing platform based on dual-emitting electrospun MOF@[Ru(byp)3]2+ nanofibers for effective detection and adsorption of aflatoxin B1

A portable, sensitive detector for the rapid detection of aflatoxin B1 (AFB1) without using biomass has been a research focal point. However, the previously documented methods were seldom applied for the simultaneous detection and elimination of AFB1 in food. In this work, the blue emission of a metal–organic framework (MOF) was selected as the fluorescence response signal, and the red fluorescence of [Ru(bpy)3]2+ was used as the reference signal. A dual-mode electrospun nanofibers ratiometric fluorescence probe (MOF@[Ru(bpy)3]2+/PAN NFs) was constructed for the first time for the quantitative detection and adsorption of AFB1. Due to the large surface area of the porous PAN NFs, these could transport MOF@Ru. AFB1 exposure led to noticeable color shift from red to blue through electron transfer. The good linear ranged of the probe for AFB1 detection of 0.0–40.0 μM and a low limit of detection of 3.05 ppb. Smartphone-integrated ratiometric sensing platform with a 5.51 ppb limit of detection was developed based on the fluorescent color change of electrospun film with AFB1. The probe was used to detect and adsorb AFB1 in peanut samples, and low relative standard deviation and satisfactory recovery rates were realized. Futher, by this speedy process, around 60 % of AFB1 could be eliminated within 30 min. Thus, the synergistic detection and removal of AFB1 is realized by using porous fluorescent MOF@Ru/PAN NFs. Moreover, the smartphone-based platform open new avenues for the on-site rapid visual quantitative detection and removal of toxins, and this is important for food security and environmental conservation applications.

Dual-readout colorimetric immunoassay based on halogenated indophenol for rapid detection of aflatoxin B1

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins. In view of the great harm of AFB1, it has attracted much attention to explore a super-sensitive and low-cost rapid detection method for aflatoxin B1. Herein, a dual-readout colorimetric immunoassay was established for the detection of AFB1. Under the catalysis of alkaline phosphatase (ALP), phenyl phosphate (PP) can produce phenol by dephosphorylation. Then phenol can react with 2,6-dibromoquinone-4-chloroimide (DBQC) under alkaline conditions to produce blue halogenated indophenol (HIP). Through this reaction, the color of the solution became blue, which can be observed by the naked eye. In addition, this color signal can be converted into digital signal via smartphone, enabling quantitative detection of AFB1. Under the optimal condition, the detection limit of this colorimetric immunoassay was 0.70 ng/mL by naked eye and 0.013 ng/mL by smartphone. The application results showed that the proposed daul-readout colorimetric immunoassay was feasible and real-time for AFB1 detection in actual samples.

Rapid detection of aflatoxin B1, zearalenone and ochratoxin A in grains by thermal desorption dielectric barrier discharge ionization mass spectrometry.

Dielectric barrier discharge ionization is increasingly used for rapid detection in ambient mass spectrometry, although more often for gaseous and highly volatile samples than for solids and liquids. In this project, we present a rapid and sensitive method for detecting mycotoxins and demonstrate its capability for the detection of aflatoxin B1, zearalenone, and ochratoxin A in food samples. Our method is based on thermal desorption coupled to dielectric barrier discharge ionization mass spectrometry (TD-DBDI-MS), which we show generates minimal interferences and produces almost exclusively molecular ions. We detected mycotoxins in various food samples, including corn, peanuts, millet, and rice. Our method has a linear dynamic range of 1 μg kg-1 to 100 μg kg-1 for all three mycotoxins and a limit of detection (LOD) of 0.31 μg kg-1, 0.28 μg kg-1 and 0.43 μg kg-1, respectively. It is simple, rapid, reduces the pretreatment steps and has significant potential for practical applications.

3D plasmonic SERS aptasensor for rapid detection of aflatoxin B1 combined with Au@Ag bimetallic nanostars and Fe3O4@MoS2 magnetic nanoflowers

As a virulent metabolite, aflatoxin B1 (AFB1) presented in various cereal grain is tightly implicated in severe human diseases. In this study, 3D plasmonic nanohybirds of Raman molecule 4-mercaptobenzoic acid (4-MBA)-embedded and AFB1 aptamer-modified bimetallic nanostars as SERS probes bound to magnetic nanoflowers were fabricated and demonstrated as a high-performance SERS-active aptasensor to quantitatively analyze AFB1. Bimetallic Au@Ag SERS plasmonic nanoprobes with enhanced properties were capable of enhancing discriminative Raman peaks of 4-MBA. Then, the integration of iron tetroxide nanoparticles (Fe3O4 NPs) and molybdenum disulfide nanosheets (MoS2 NShs) with huge specific surface area constituted stable 3D Fe3O4@MoS2 plasmonic nanoflowers, facilitating the bind of numerous aptamer-based SERS probes via the non-covalent interaction between MoS2 NShs and aptamer, which were ideal candidates for SERS-active substrates. Additionally, Fe3O4 NPs as magnetic core endowed 3D nanocomposites with specific magnetic separation characteristic that caused the collected SERS hotspots to exhibit superior signal response, and further strengthening the sensitivity in a complex food matrix. Aptamer-target AFB1 specific recognition triggered linearly diminished 4-MBA signal intensity (I4-MBA) on the substrate to achieve a low detection limit of 58.9 pg/mL. Furthermore, the sensor has the potential to be a promising monitoring tool for other trace contaminants.

Development of a Rapid Gold Nanoflowers Immunochromatographic Test Strip Based on the Nanobody for Detection of Aflatoxin B1

AbstractA rapid gold nanoflowers (AuNFs) immunochromatographic test strip (ICTS) based on the nanobody (Nb) was developed and used for rapid detection of Aflatoxin B1 (AFB1). Thanks to remarkable physicochemical stability, structural adaptability, ease of genetic manipulation, expression in the prokaryotic system with high yield and tolerance to harsh environments of nanobodies, the G8‐DIG was produced via expression and purification, which was labeled with AuNFs by electrostatic adsorption as signal probes in the ICTS. The AuNFs‐anti‐G8‐DIG nanobodies as a probe of ICTS could specifically recognize not only AFB1 but also DIG‐BSA. In this study, the DIG‐BSA replaced the anti‐6×His‐tag antibody of traditional nanobody‐based ICTS was coated on the control line, which was easier to synthesize, cheaper, and more stable. Under the optimal conditions, the AuNFs‐based nanobody ICTS system accurately detected AFB1 linearly and dynamically over the range (IC20~IC80) of 1.02~27.86 ng/mL, with the IC10 and IC50 of 0.1 ng/mL and 5.46 ng/mL respectively. In addition, these developed nanobodies ICTS had a universal application in the analysis of AFB1 in corn samples.

An effective strategy of designing “turn on” mode-based lateral flow chromatography for rapid detection of aflatoxin B1

Aflatoxin B1 (AFB1) is an extremely toxic food contaminant that has serious effects on human health. In published aptamer-based lateral flow chromatography test strips, the detection results are mostly inversely proportional to the concentration of AFB1, which is a serious violation of intuition. An aptamer sensor based on the “turn on” mode for AFB1 detection has been reported in this paper. In this paper, we use gold nanoparticles (AuNPs) to present colorimetric signals and link capture sequences (SH-poly A-cDNA) to design AuNPs@SH-poly A-cDNA nanoprobes. The test strip relies on AFB1 and AuNPs@SH-poly A-cDNA competing aptamers for rapid detection of AFB1. In the absence of AFB1, the aptamer binds to AuNPs@SH-poly A-cDNA, preventing the cDNA from binding to the complementary fragment of the test line (DNAT), resulting in the disappearance of the red signal. However, in the presence of AFB1, the aptamer binds to AFB1, allowing some AuNPs@SH-poly A-cDNA not bound to the aptamer to be captured by DNAT, leading to the generation of a red signal. Regardless of the presence or absence of AFB1, the poly-A fragment of AuNPs@SH-poly A-cDNA consistently binds to the complementary sequence of the control line (DNAC) and presents a red signal. The detection results are proportional to the concentration of AFB1, which is in line with human intuitions. The development of this test strip provides a novel design idea for the detection of small molecules such as mycotoxins.

A novel fluorescent sensing platform based on nitrogen-doped carbon quantum dots for rapid and sensitive detection of aflatoxin B1 in corn flour

Aflatoxin B1 (AFB1) contamination in cereals and their products seriously concerns food safety and human health. Herein, a nitrogen-doped carbon quantum dots (N-CQDs)-based fluorescent sensing platform was successfully developed for the sensitive and rapid detection of AFB1 in corn flour. In this work, using sodium citrate and urea as precursors, hydrophilic N-CQDs were synthesized by the one-step hydrothermal method with good biocompatibility and high fluorescence stability. The as-prepared N-CQDs exhibited excitation wavelength-independent fluorescence in the 300–370 nm range, and had bright blue emission at 448 nm upon 360 nm excitation. Via fluorescence quenching of the N-CQDs in the presence of increasing AFB1 concentrations, the proposed fluorescent sensor had an excellent selectivity for AFB1 detection with a low limit of detection (0.13 ng/mL) in the linear range of 0.5–100 ng/mL. Under optimized sensing conditions, the rapid AFB1 determination in real corn flour samples was realized with good recoveries (95.3–105.7%). In summary, present results indicated the promising potential of this novel N-CQDs-based fluorescent sensor for rapid and effective AFB1 detection in agricultural products.

"Green" Extraction and On-Site Rapid Detection of Aflatoxin B1, Zearalenone and Deoxynivalenol in Corn, Rice and Peanut.

The common mycotoxins in polluted grains are aflatoxin B1(AFB1), zearalenone (ZEN) and deoxynivalenol (DON). Because of the potential threat to humans and animals, it is necessary to detect mycotoxin contaminants rapidly. At present, later flow immunoassay (LFIA) is one of the most frequently used methods for rapid analysis. However, multistep sample pretreatment processes and organic solvents are also required to extract mycotoxins from grains. In this study, we developed a one-step and "green" sample pretreatment method without using organic solvents. By combining with LFIA test strips and a handheld detection device, an on-site method for the rapid detection of AFB1, ZEN and DON was developed. The LODs for AFB1, ZEN and DON in corn are 0.90 μg/kg, 7.11 μg/kg and 10.6 μg/kg, respectively, and the working ranges are from 1.25 μg/kg to 40 μg/kg, 20 μg/kg to 2000 μg/kg and 35 μg/kg to 1500 μg/kg, respectively. This method has been successfully applied to the detection of AFB1, ZEN and DON in corn, rice and peanut, with recoveries of 89 ± 3%-106 ± 3%, 86 ± 2%-108 ± 7% and 90 ± 2%-106 ± 10%, respectively. The detection results for the AFB1, ZEN and DON residues in certified reference materials by this method were in good agreement with their certificate values.

High performance aptasensing platform development through in silico aptamer engineering for aflatoxin B1 monitoring

Due to the technical challenges of small binding aptamer development, reliable computational simulation studies can be considered as effective tools to design novel and high functional mycotoxin aptameric probes. Here, two novel aflatoxin B1(AFB1) binding aptamers were successfully exploited as recognition elements in the lateral flow aptasensors and the reflective phantom interface (RPI) platform. Using the parent aptamer previously designed through genetic algorithm based in silico maturation (ISM) strategy, F20, a new variant, F20-T, was obtained here via coupling truncating strategy and computational simulation approaches. Two aptamer-gold nanoparticle strip biosensors were developed based on the designed probes for the simple and rapid detection of AFB1 in competitive format. The F20-based strip was more sensitive than that exploiting the truncated aptamer, with limits of detection (LOD) of 0.1 and 0.5 ng/mL, respectively. Based on the in silico and experimental selectivity evaluations of both test strips towards other mycotoxins, including aflatoxin B 2 , M 1 , G 1 , G 2 , Ochratoxin A and Zearalenone, F20-T based test strip revealed higher selectivity for AFB1. Both developed aptasensors successfully detected AFB1 in maize flour within 30 min using a simple strip reader. Exploiting of F20 and F20-T aptamers in an exclusive technology called RPI platform led to successful AFB1 detection, as well. Both designed aptameric probes can be regarded as potential recognition elements to develop screening tools for rapid, low cost and on-site AFB1 detection. Our findings highlighted the reliable and robust application of computational simulation studies for novel small binding aptamer development and consequently open up a much-needed avenue to design various aptasensing platforms in green and cost effective ways. • A new truncated Aflatoxin B1 binding aptamer was designed via in silico studies. • The truncated and the parent aptamer were successfully applied to build aptasensors. • The redesigned aptasensing platforms provided high sensitive AFB1 detection. • In silico engineered aptamers can be costly exploited for new aptasensor development.

Complementary DNA Significantly Enhancing Signal Response and Sensitivity of a Molecular Beacon Probe to Aflatoxin B1

This paper reported an improved molecular beacon method for the rapid detection of aflatoxin B1 (AFB1), a natural mycotoxin with severe carcinogenicity. With the assistance of a complementary DNA (cDNA) chain, the molecular beacon which consists of a DNA aptamer flanked by FAM and BHQ1 displayed a larger fluorescent response to AFB1, contributing to the sensitive detection of AFB1. Upon optimization of some key experimental factors, rapid detection of AFB1 ranging from 1 nM to 3 μM, within 20 min, was realized by using this method. A limit of detection (LoD) of 1 nM was obtained, which was lower than the LoD (8 nM) obtained without cDNA assistance. This aptamer-based molecular beacon detection method showed advantages in easy operation, rapid analysis and larger signal response. Good specificity and anti-interference ability were demonstrated. This method showed potential in real-sample analysis.

A SERS aptasensor for rapid detection of aflatoxin B1 in coix seed using satellite structured Fe3O4@Au nanocomposites

Coix seed is a minor cereal that has been used as a functional food in the Chinese diet due to its high nutritional value and health effects. These claimed health benefits include antioxidation, anti-cancer, anti-inflammation and enhancing immunological activity. But coix seed is easily contaminated by various mycotoxin, of which aflatoxin B 1 (AFB 1 ) is one of major threat to human and animal health. For the sake of detecting AFB 1 more effectively, a surface enhanced Raman spectroscopy (SERS) based aptasensor for rapid detection of AFB 1 in coix seed was developed. Fe 3 O 4 @Au nanoparticles (NPs) modified with aptamer which possess the ability of magnetic separation and excellent SERS activity were synthesized and characterized by transmission electron microscope. Ag NPs modified with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and complementary DNA (cDNA) were employed as SERS tags. Controllable hot spots were built by the combination of Fe 3 O 4 @Au NPs and Ag NPs through AFB 1 aptamer and cDNA, and also by the interparticle nanogapped between the different size of Au NPs. Based on the principle of competitive binding between AFB 1 and cDNA for aptamer, the rapid quantitative detection of AFB 1 in coix seed was realized. The limit of detection (LOD) was determined to be 0.0060 ng/mL, and a broad linear range between 0.01 and 100 ng/mL was achieved. The spiked recovery rates of coix seed sample were 98.2%–101.2%. The results demonstrated the promising potential of this aptasensor in rapid and effective detection of AFB 1 in foodstuffs. • A SERS aptasensor for the rapid detection of AFB 1 in coix seed was constructed. • Nanogapped Au NPs can provide a lot of controllable hot spots. • GMNPs modified with aptamer exhibited enrichment ability and excellent SERS activity.

A High-Performance Self-Supporting Electrochemical Biosensor to Detect Aflatoxin B1.

High-performance electrochemical biosensors for the rapid detection of aflatoxin B1 (AFB1) are urgently required in the food industry. Herein, a multi-scaled electrochemical biosensor was fabricated by assembling carboxylated polystyrene nanospheres, an aptamer and horseradish peroxidase into a free-standing carbon nanofiber/carbon felt support. The resulting electrochemical biosensor possessed an exceptional performance, owing to the unique structures as well as the synergistic effects of the components. The 3D porous carbon nanofiber/carbon felt support served as an ideal substrate, owing to the excellent conductivity and facile diffusion of the reactants. The integration of carboxylated polystyrene nanospheres with horseradish peroxidase was employed as a signal amplification probe to enhance the electrochemical responses via catalyzing the decomposition of hydrogen peroxide. With the aid of the aptamer, the prepared sensors could quantitatively detect AFB1 in wine and soy sauce samples via differential pulse voltammetry. The recovery rates of AFB1 in the samples were between 87.53% and 106.71%. The limit of detection of the biosensors was 0.016 pg mL−1. The electrochemical biosensors also had excellent sensitivity, reproducibility, specificity and stability. The synthetic strategy reported in this work could pave a new route to fabricate high-performance electrochemical biosensors for the detection of mycotoxins.

Pixel-level rapid detection of aflatoxin B1 based on 1D-modified temporal convolutional network and hyperspectral imaging

Aflatoxin is a virulent toxic and cancerogenic substance, and it is widespread on the peanuts surface. This study uses a one-dimensional modified temporal convolutional network (1D-modified TCN) to detect aflatoxins at the pixel level with hyperspectral images. Then, the robustness of this method was verified from compressed image size and reduced spectral acquisition points, and feasible strategies were provided for accelerating model optimization training. The experimental results indicate that 1D-modified TCN is best under the image size of 63×47, the model training accuracy was 99.60 %, and the test accuracy was 99.26 %. Finally, the detection results of the four one-dimensional network models were visualized on three new peanut kernels. This method has improved the accuracy of aflatoxin detection and it will quickly promote the design and development of intelligent devices in detecting aflatoxin.

Nanobody-Based Lateral Flow Immunoassay for the Rapid Detection of Aflatoxin B1 in Almond Milk

Aflatoxin B1 (AFB1) is the most potent genotoxic and carcinogenic aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus. AFB1 is considered a major concern in food safety because of its high toxicity and possible contamination of a variety of food commodities such as cereals, groundnuts, tree nuts, and milk. To guarantee the food safety and address health concerns of consumers, it is important to monitor the contamination of the food chain with this toxin. Screening and regular testing for aflatoxin are mainly done by reference laboratories from the European Union (EU) and the United States (US), using sensitive but low-throughput technologies, thus limiting the frequency of product testing. The problem is still more serious in developing countries, where contamination is often high and rapid field methods are needed. New technologies are needed to provide broader, more thorough and efficient screening for these mycotoxins. In this context, we present the development of a lateral flow immunoassay (LFIA) for the detection of AFB1 as a semiquantitative approach. We have combined the advantages of engineered nanobodies specific to AFB1 with an easy fluorescence read-out system of green fluorescence protein (GFP) fused as a label to a specific nanobody. Optimized LFIA reached a limit of detection (LOD) of 4.80 ng/mL in undiluted almond milk. Because of lifestyle changes, there is an increasing demand for almond milk compared to dairy products. Almond milk is susceptible to contamination with aflatoxin and thus was selected as a food matrix for AFB1 monitoring. The U.S. Food and Drug Administration (FDA) and the European Commission established an action level or maximum residue level of 20 and 8 ng/mL for almond milk, respectively. The developed LFIA has been optimized to distinguish between compliant/noncompliant samples taking into consideration the signal provided by the control line (CL). The CL signal has been set as a threshold of the maximum residue level (MRL) signal (TL). A validation study has been performed and blind samples have been measured successfully.

Electrochemical immunosensor based on AuNPs/Zn/Ni-ZIF-8-800@graphene for rapid detection of aflatoxin B1 in peanut oil

Peanut oil is a basic food raw material in life. However, aflatoxin contamination in peanut oil is considered to be one of the most serious food safety problems in the world. Based on AuNPs/Zn/Ni-ZIF-8-800@graphene composite, a simple, efficient and sensitive electrochemical immunosensor was developed to detect aflatoxin B1 (AFB1) in peanut oil. The bare glassy carbon electrode was modified by graphene, bimetallic organic framework material (Zn/Ni-ZIF-8-800), chitosan and gold nanoparticles. The electrochemical immunosensor was characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) and cyclic voltammetry (CV), and the electrochemical signal changes after antibody and AFB1 binding were investigated in detail. Under the optimal conditions, the linear range of the electrochemical immunosensor was 0.18–100 ng/mL, and the detection limit was 0.18 ng/mL. In addition, the prepared sensor has high selectivity and long-term stability, which lays a foundation for the simple, rapid and sensitive detection of AFB1 in peanut oil.

An ultralow concentration of Al-MOFs for turn-on fluorescence detection of aflatoxin B1 in tea samples

A turn-on fluorescent sensing platform based on an ultralow concentration of Al-metal organic frameworks for the detection of aflatoxin B1 has been developed for the first time. This fluorescence turn-on sensor exhibits the largest fluorescence enhancement (or quenching) constant value of 179404 M−1 among all luminescence-based chemical sensors reported till date. Moreover, the sensor afforded a rapid detection of aflatoxin B1, with a linear response in the concentration range of 0.05–9.61 μM and a low detection limit of 11.67 ppb. Additionally, the fabricated sensor showed good repeatability, reproducibility, stability, and selectivity. Most importantly, the practical application of this sensor has been demonstrated by detecting aflatoxin B1 in complex tea samples with low relative standard deviation (≤7.72%; n = 3) and satisfactory recoveries. In summary, the proposed method has great potential as a simple, sensitive and selective strategy for monitoring aflatoxin B1 in food samples.

Rapid and label-free detection of aflatoxin B1 using a rationally truncated aptamer and via circular dichroism measurement

We describe a simple aptamer assay for the rapid analysis of aflatoxin B1 (AFB1), a potently carcinogenic fungal metabolite. The original anti-AFB1 aptamer was rationally truncated, obtaining a short aptamer that displayed better performance. Different concentrations of AFB1 were detected by measuring the circular dichroism spectra of this short aptamer. The detection limit reached 0.6 nM, which was lower than that of the original aptamer (80 nM).

Ultrafast Ratiometric Detection of Aflatoxin B1 Based on Fluorescent β-CD@Cu Nanoparticles and Pt2+ Ions.

Rapid detection of aflatoxin B1 (AFB1) is a very important task in food safety monitoring. However, it is still challenging to achieve highly sensitive detection without antibody or aptamer biomolecules. In this work, a rapid detection of aflatoxin B1 was achieved using a ratiometric fluorescence probe without antibody or aptamer for the first time. In the ratiometric fluorescence system, the fluorescence emission of AFB1 at 433 nm was significantly enhanced due to the β-cyclodextrin-AFB1 host-guest interaction and the complexation of AFB1 and Pt2+. Meanwhile, the inclusion of aflatoxin B1 also quenched the fluorescence emission of β-CD@Cu nanoparticles (NPs) at 650 nm based on inner filter effect mechanism. On the basis of the above effects, the ratiometric detection of aflatoxin B1 was achieved in the range of 0.03-10 ng/mL with a low detection limit of 0.012 ng/mL (3σ/s). In addition, the β-CD@Cu NPs based nanoprobe could achieve stable response within 1 min to AFB1. The above ratiometric detection also demonstrated excellent application potential in the rapid on-site detection of AFB1 in food due to the advantages of convenience, rapidness, and high accuracy.

Development of a biotinylated nanobody for sensitive detection of aflatoxin B1 in cereal via ELISA

Aflatoxin B1 (AFB1) contamination is a severe threat to food safety and human health, and requires continuous monitoring. In this study, we developed a biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) by using biotinylated nanobody Nb26 and streptavidin-conjugated polymerized horseradish peroxide (SA-PolyHRP) for sensitive and rapid detection of AFB1 in cereal. Under the optimal condition, the IC50 value of the BA-ELISA was improved to 0.21 ng mL−1 for AFB1, satisfying the requirement of detection limit in practical applications. The total assay time of our strategy is reduced to 50 min from 2 h in conventional competitive ELISA. Additionally, the BA-ELISA saves as much as 98% of the antibody in comparison to the previous classic ELISA. Our work also demonstrated an interesting phenomenon that the biotinylated Nb26 achieved better selectivity to AFB1, which could possibly result from the steric hindrance that interferes reaction between the Nb26 and the AFB1 analogs. Furthermore, the assay was used to detect AFB1 in two cereal samples, and the results were in good agreement with that obtained by high performance liquid chromatography. The developed BA-ELISA can be used for routine screening analysis of AFB1, and offers a promising strategy for measuring low concentrations of food contaminants.