Sensitive Detection Of Aflatoxin B1 Research Articles

A Simple Electrochemical Immunosensor for Highly Sensitive Detection of Aflatoxin B1 Based on Gold Nanoparticle Decorated Carboxylated Graphene Oxide

A Simple Electrochemical Immunosensor for Highly Sensitive Detection of Aflatoxin B1 Based on Gold Nanoparticle Decorated Carboxylated Graphene Oxide

Fluorometric determination of aflatoxin B1 using a labeled aptamer and gold nanoparticles modified with a complementary sequence acting as a quencher.

A fluorometric aptamer based assay is described for rapid and sensitive detection of aflatoxin B1 (AFB1). It is making use of a fluorescein (FAM) labeled anti-AFB1 aptamer and complementary DNA-modified gold nanoparticles (GNPs). In the absence of AFB1, the FAM-labeled aptamers hybridize with complementary DNA strands that were covalently immobilized on GNPs. This results in quenching of the green fluorescence (with excitation/emission peaks at 485/525nm). In the presence of AFB1, the aptamer probe binds AFB1 and is released from the GNPs. Hence, fluorescence is restored. Under optimized conditions, AFB1 in the concentration range from 61 pM to 4.0μM can be detected, and the detection limit is 61 pM. This assay is highly selective for AFB1. It was applied to the determination of AFB1 spiked into 50-fold diluted wine and 20-fold diluted beer. Graphical abstract Schematic presentation of fluorometric detection of AFB1 using a fluorescein (FAM) labeled anti-AFB1 aptamer and complementary DNA-modified gold nanoparticles (GNPs).

A signal-on electrochemical aptasensor for rapid detection of aflatoxin B1 based on competition with complementary DNA

Aflatoxin B1 (AFB1) is the most toxic mycotoxin, causing harmful effects on human and animal health, and the rapid and sensitive detection of AFB1 is highly demanded. We developed a simple electrochemical aptasensor achieving rapid detection of aflatoxin B1 (AFB1). A short anti-AFB1 aptamer having a methylene blue (MB) redox tag at the 3′-end was immobilized on the surface of a gold electrode. In the absence of AFB1, a complementary DNA (cDNA) strand hybridized with the MB-labeled aptamer, causing MB apart from the electrode surface and low current of MB. In the presence of AFB1, AFB1 competed with the cDNA in the binding to the MB-labeled aptamer, and the aptamer-AFB1 binding caused formation of a hairpin structure, making the MB close to the electrode surface and current of MB increase. Under optimized conditions, we achieved detection of AFB1 over dynamic concentration range of 2 nM–4 μM by using this signal-on electrochemical aptasensor. This method only required a simple 5-min incubation of sample solution prior to rapid electrochemical sensing, more rapid than other electrochemical aptasensors. The sensor could be well regenerated and reused. This sensor allowed to detect AFB1 spiked in 20-fold diluted beer and 50-fold diluted white wine, respectively. It shows potential for detection of AFB1 in wide applications.

Single-Particle LRET Aptasensor for the Sensitive Detection of Aflatoxin B1 with Upconversion Nanoparticles.

Contamination of foods and feeds by aflatoxins is a universal yet serious problem all over the world. Particularly, aflatoxin B1 (AFB1) is the most primary form and readily leads to terrible damages to human health. In this work, we construct a sensitive aptasensor based on single-particle detection (SPD) to analyze AFB1 in peanut samples with luminescence resonance energy transfer (LRET) between the aptamer-modified upconversion nanoparticles (UCNPs-aptamer) and gold nanoparticles (GNPs). The UCNP-aptamer plays as the luminescence donor, while GNP acts as the energy acceptor. In the absence of AFB1, GNPs would adsorb onto the surface of UCNPs-aptamer because of the association between aptamers and GNPs, leading to luminescence quenching. However, the luminescence of UCNPs-aptamer is recovered gradually in the presence of AFB1, because the aptamers possess stronger affinity toward AFB1 than GNPs. Through statistically counting the number of luminescent particles on the glass slide surface, the concentration of AFB1 in solution is accurately determined. The linear dynamic range for AFB1 detection is from 3.13 to 125.00 ng/mL. The limit-of-detection (LOD) is 0.17 ng/mL, which is much lower than the allowable concentration in foods. As a result, this method would provide promising application for the sensitive detection of AFB1 in foods and feeds, which might make a meaningful contribution to food safety and public health in the future.

Label-free impedimetric immunosensors for sensitive detection of aflatoxin B1 in food

Aflatoxin B1 (AFB1) is one of the mycotoxins highly resistant to food processing that enters the food chain and provide a threat to human health. In this paper, different immobilization techniques of the monoclonal anti-AFB1 on gold electrodes were tested and compared, in order to develop a label-free immunosensor able to detect AFB1 amount imposed by European legislation for adult and infant foods. Different materials, coupled with Electrochemical Impedance Spectroscopy as transduction technique, were used to improve immunosensors analytical parameters, such as linear range, sensitivity and limit of detection. Through the use of ferrocene molecules, the immunosensor showed linearity in the range 0.01-10 ng/mL and the lowest limit of detection (0.01 ng/mL), allowing the possibility to use it in a wide range of food products, including infant foods.

A novel fluorescence aptasensor based on mesoporous silica nanoparticles for selective and sensitive detection of aflatoxin B1

Based on the mesoporous silica nanoparticles (MSN), a novel, simple and label-free aptamer biosensor was designed for the detection of aflatoxin B1 (AFB1). Here, the aptamers were used as molecular recognition probes and “gated molecules” while Rh6G was loaded into the interior of the particles as the signal probe. In the absence of AFB1, the “gate” was closed to prevent the leakage of the signal probe because of the immobilization of aptamers on the surface of MSN-NH2. With the presence of AFB1, the “gate” could be opened to release the signal probe for the specifical binding of aptamers to AFB1. Our results showed that the fluorescence intensity was positively correlated with the concentration of AFB1 (0.5–50 ng mL−1), with the detection limit as low as 0.13 ng mL−1. What's more, this design provides a new approach for rapid, sensitive and selective detection based on aptamers and it could be applied to numerous other analytes if appropriate aptamers are available.

Fabricating photoelectrochemical aptasensor for sensitive detection of aflatoxin B1 with visible-light-driven BiOBr/nitrogen-doped graphene nanoribbons

A selective and sensitive photoelectrochemical (PEC) aptasensor for AFB1 detection in corn samples was fabricated by introducing BiOBr/nitrogen-doped graphene nanoribbons (N-GNRs) as photoactive interface. As efficient visible-light-driven photoactive species, the prepared BiOBr/N-GNRs exhibited higher photoactivity than pure materials under visible light irradiation. N-GNRs, acting as promising supporters for nanomaterials, can improve the performance of composites. UV–vis diffuse reflectance spectroscopy demonstrated that BiOBr/N-GNRs possessed the narrower band gap energy, which could be easily irradiated by visible light. In addition, steady-state photoluminescence (PL) spectra also revealed that BiOBr/N-GNRs exhibited the lower recombination rate of photogenerated electron−hole pairs. The formation of the aptamer-AFB1 complex increased the resistance of the electrode, restrained the electron transfer, and thus quenched the PEC signal. Thereafter, a “signal-off” PEC aptasensor was fabricated successfully. The proposed PEC aptasensor demonstrated sensitive detection of AFB1 in a wide range from 5pgmL−1 to 15ngmL−1 with a low detection limit of 1.7pgmL−1 and possessed high specificity and good reproducibility. The PEC aptasensor was suitable for corn samples with a good recovery in the range of 98.0–102.0% and the relative standard deviation (RSD) of 1.7–2.1% to confirm practical utility. Furthermore, this strategy would be extended to detect different targets as versatile PEC devices by replacing the aptamers with other sequences.

A rapid fluorometric method for determination of aflatoxin B1 in plant-derived food by using a thioflavin T-based aptasensor

A fluorometric aptamer-based method is described for the determination of aflatoxin B1 (AFB1). The fluorescent dye thioflavin T (ThT) forms a complex with the aptameragainst AFB1 (aptamer/ThT), and the fluorescence of the complex is strongly enhanced. On addition of AFB1, it will bind to the aptamer and release ThT. The fluorescence of free ThT is much weaker. The fluorescence of the system, best measured at excitation/emission wavelengths of 440/487nm, drops gradually in the AFB1 concentration range from 0.2 to 200ng·mL-1, exhibiting good linearity. The detection limits are 0.2ng·mL-1 in buffer solution, and 1ng·mL-1 when applied to plant-derived food. The recovery of AFB1 from spiked foodstuff ranges from 74.7% to 121%. The assay can be performed within 20min. Graphical abstract Schematic presentation of label-free thioflavin T (ThT)-based fluorescence aptasensor using aflatoxin B1(AFB1) aptamer/ThT G-quadruplex complex and the AFB1 aptamer/AFB1 complex fluorescent signal system for the rapid and sensitive detection of AFB1 in soy sauce,spirits, rice, corn and peanuts.

A simple aptamer molecular beacon assay for rapid detection of aflatoxin B1

Aflatoxin B1 (AFB1) is a highly toxic mycotoxin, and rapid and sensitive detection of AFB1 is in demand for food safety and environmental analysis. Here we described a simple aptamer molecular beacon assay for rapid detection of aflatoxin B1 (AFB1) by using an aptamer with a fluorescein (FAM) label at the 5′ end and a fluorescence quencher (black hole quencher 1, BHQ1) at the 3′ end. In the presence of AFB1, the aptamer probe bound with AFB1 and induced a hairpin structure, drawing FAM and BHQ1 into close proximity and leading to fluorescence quenching. This assay allowed for a detection limit of 3.9 nmol/L and a dynamic range from 3.9 nmol/L to 4 μmol/L. Specificity test showed other mycotoxins including ochratoxin A, ochratoxin B, fumonisin B1, fumonisin B2, and zearalenone had negligible influence on detection of AFB1. AFB1 spiked in diluted liquor wine, methanol, or corn flour samples was successfully detected by using this aptamer probe, and the assay showed potential for real sample analysis.

A photoelectrochemical aptasensor for aflatoxin B1 detection based on an energy transfer strategy between Ce-TiO2@MoSe2 and Au nanoparticles.

In this work, a novel photoelectrochemical (PEC) aptasensor was developed for the sensitive detection of aflatoxin B1 (AFB1) based on a resonance energy transfer strategy between the Ce-TiO2@MoSe2 heterostructure and Au nanoparticles (AuNPs). The Ce-TiO2@MoSe2 composite was obtained by growing MoSe2 nanosheets on a TiO2 nanocube doped by the Ce element with a facile hydrothermal method. The composite effectively extended the absorption of TiO2 to the visible region and avoided the self-aggregation of MoSe2 nanosheets, leading to the excellent photocurrent response under visible light excitation. The PEC aptasensor was then fabricated by immobilizing the Ce-TiO2@MoSe2 composite on an ITO electrode, followed by the modification of the aminated AFB1 aptamer. An AuNP-labeled DNA sequence was subsequently hybridized with the aptamer to fabricate a sandwich structure, which was destroyed after the introduction of AFB1, decreasing the amount of the energy acceptor (AuNPs) at the electrode surface. Accordingly, the photocurrent was increased with the increase of AFB1 concentration. Under the optimal conditions, the PEC aptasensor showed a wide linear range of 0.03-200 ng mL-1 and a low detection limit of 0.01 ng mL-1 for AFB1 determination.

Etching reaction-based photoelectrochemical immunoassay of aflatoxin B1 in foodstuff using cobalt oxyhydroxide nanosheets-coating cadmium sulfide nanoparticles as the signal tags

A new split-type photoelectrochemical (PEC) immunosensing platform was designed for sensitive detection of aflatoxin B1 (AFB1) in foodstuffs, coupling with enzymatic hydrolysate-triggered etching reaction of cobalt oxyhydroxide (CoOOH) on cadmium sulfide (CdS) nanoparticles-functionalized interface. Initially, the photosensitive electrode was prepared by coating CoOOH nanosheets on the surface of CdS nanoparticles to quench the photocurrent. Thereafter, a competitive-type enzyme immunoreaction was carried out on monoclonal anti-AFB1 antibody-conjugated magnetic bead by using alkaline phosphatase (ALP)-labeled bovine serum albumin-AFB1 (AFB1-BSA) conjugate as the competitor. With the formation of immunocomplex, the carried ALP hydrolyzed ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA) and phosphate. The former ascorbic acid produced etched or dissolved CoOOH nanosheets into Co2+ ions, thus resulting in the exposure of CdS nanoparticles on the surface to enhance the photocurrent of the modified electrode. Under optimum conditions, the photocurrent decreased linearly with the increasing AFB1 concentration in the dynamic range of 0.01–10 ng mL−1, and the limit of detection was 2.6 pg mL−1. The precision of this method (expressed as RSD) was ±8.6%. In addition, the accuracy was monitored by analyzing spiked food samples, and gave the well-matched results with the referenced ELISA method.

Inner-filter effect based fluorescence-quenching immunochromotographic assay for sensitive detection of aflatoxin B1 in soybean sauce

A fluorescence-quenching immunochromotographic assay (ICA) was developed for sensitive detection of aflatoxin B1 (AFB1) in soybean sauce based on the inner filter effect (IFE) between flower-like gold nanoparticles (AuNFs) and quantum dots (QDs). QDs were sprayed on the test and control line zones as background fluorescence signals, whereas AuNFs were designed as the fluorescence absorber of QDs because the surface plasma resonance peak of AuNFs totally matched with the maximum emission peak of QDs. Under the optimal conditions, the fluorescence-quenching ICA strip showed a good linear detection for AFB1 in standard AFB1 solution from 0.008 μg/L to 1 μg/L with a low detection limit of 0.004 μg/L. The average recoveries for different concentrations of AFB1-spiked soybean sauce samples ranged from 84.69% to 120.44% with a coefficient of variation ranging from 2.73% to 10.41%. In addition, the reliability of the proposed method was further confirmed by ultra-performance liquid chromatography with fluorescence detection method. In brief, this novel IFE-based strip offers a simple, rapid, sensitive, and accurate strategy for quantitative detection of AFB1 in soybean sauce.

A new amplified fluorescent aptasensor based on hairpin structure of G-quadruplex oligonucleotide-Aptamer chimera and silica nanoparticles for sensitive detection of aflatoxin B1 in the grape juice

As one of the most toxic mycotoxins, aflatoxin B1 (AFB1) is a major food pollutant which can pose a high risk to human health. In this work, an accurate fluorescent sensing method was proposed for AFB1 determination, based on hairpin structure of G-quadruplex oligonucleotide-Aptamer chimera, silica nanoparticles coated with streptavidin (SNPs-Streptavidin) and N-methyl mesoporphyrin IX (NMM). The hairpin structure of chimera and SNPs-Streptavidin allowed AFB1 detection with high sensitivity and specificity. Moreover, the developed sensor could detect AFB1 in 30 min. The relative fluorescence intensity increased as AFB1 concentrations increased with a linear range of 30–900 pg/mL and a limit of detection (LOD) of 8 pg/mL. The constructed aptasensor was successfully employed to assess AFB1 spiked grape juice and human serum samples. The analytical recovery of AFB1 in the grape juice samples ranged from 95 to 106%, implying the great potential of the presented aptasensor in food product analysis.

Plasmonic ELISA based on enzyme-assisted etching of Au nanorods for the highly sensitive detection of aflatoxin B1 in corn samples

In this work, we reported an advanced enzyme-assisted etching method using gold nanorods (AuNRs) as signal output of plasmonic enzyme-linked immunosorbent assay (pELISA) for the quantitative or qualitative detection of aflatoxin B1 (AFB1) in real corn samples. AFB1-labeled glucose oxidase was adopted as the competing antigen to catalyze glucose into H2O2. Meanwhile, horseradish peroxidase was employed to catalyze H2O2 and hence generate .OH. The AuNRs with the aspect ratio of 2:1 were chemically etched by OH to a rod-like morphology. This occurrence reduced the optical density at 650 nm and resulted in a vivid color response from bluish-green to red easily detectable by a microplate reader for quantitative AFB1 analysis or by the naked eye for qualitative AFB1 detection. Various parameters that may influence the detection performance of pELISA were investigated. The proposed method showed an extremely high sensitivity for qualitative AFB1 detection, with a visible cut-off value of 12.5 pg/mL. The technique also achieved a good linear range of 3.1–150 pg/mL for quantitative AFB1 analysis, with a half-maximal inhibitory concentration of 22.3 pg/mL, which was approximately 32 folds lower than those of conventional ELISA (707 pg/mL). The average recoveries for corn samples spiked with different concentrations of AFB1 ranged from 82% to 115%, with a coefficient of variation that ranged from 2% to 13%. These values corresponded to an acceptable accuracy and precision for the proposed method. In addition, the reliability of the proposed method was further confirmed by the liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. In brief, this work offers an improved screening strategy with high sensitivity and robustness for the qualitative or quantitative detection of mycotoxins or other pollutants in food safety monitoring.

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.

Novel photoluminescence enzyme immunoassay based on supramolecular host-guest recognition using L-arginine/6-aza-2-thiothymine-stabilized gold nanocluster

A new photoluminescence (PL) enzyme immunoassay was designed for sensitive detection of aflatoxin B1 (AFB1) via an innovative enzyme substrate, 6-aza-2-thiothymine-stabilized gold nanocluster (AAT-AuNC) with L-arginine. The enzyme substrate with strong PL intensity was formed through supramolecular host-guest assembly between guanidine group of L-arginine and AAT capped on the surface of AuNC. Upon arginase introduction, the captured L-arginine was hydrolyzed into ornithine and urea, thus resulting in the decreasing PL intensity. Based on this principle, a novel competitive-type immunoreaction was first carried out on AFB1-bovine serum albumin (AFB1-BSA) conjugate-coated microplate, using arginase-labeled anti-AFB1 antibody as the competitor. Under the optimum conditions, the PL intensity increased with the increment of target AFB1, and allowed the detection of the analyte at concentrations as low as 3.2 pg mL−1 (ppt). Moreover, L-arginine-AAT-AuNC-based PL enzyme immunoassay afforded good reproducibility and acceptable specificity. In addition, the accuracy of this methodology, referring to commercial AFB1 ELISA kit, was evaluated to analyze naturally contaminated or spiked peanut samples, giving well-matched results between two methods, thus representing a useful scheme for practical application in quantitative monitoring of mycotoxins in foodstuff.

Amino-functionalized CdSe/ZnS quantum dot-based lateral flow immunoassay for sensitive detection of aflatoxin B1

In this study, highly fluorescent amino-functionalized CdSe/ZnS QDs coated with amphiphilic N-alkylated poly(ethyleneimine) have been synthesized and applied as fluorescent probes in LFIA strips for on-site determination of AFB1.

高灵敏磁分离荧光传感法检测黄曲酶毒素B1

高灵敏磁分离荧光传感法检测黄曲酶毒素B1

Competitive horseradish peroxidase-linked aptamer assay for sensitive detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is one of highly toxic mycotoxins and a known human carcinogen. The frequent contamination of AFB1 in food products and large health risk of AFB1 have raised global concerns. Sensitive detection of AFB1 is of vital importance and highly demanded. Herein, we reported a competitive horseradish peroxidase (HRP)-linked aptamer assay for AFB1, combining the advantages of aptamer for affinity binding and enzyme label for signal amplification. In this assay, free AFB1 in solution competed with a covalent conjugate of bovine serum albumin-AFB1 (BSA-AFB1) coated on the wells of microplate in binding to the HRP-labeled aptamer probe. HRP attached on BSA-AFB1 in the wells catalyzed the conversion of substrates into products, allowing the final detection of AFB1 through measurement of the generated products. When TMB (3,3′,5,5′-tetramethylbenzidine dihydrochloride) was used as substrate, absorbance analysis of the product of enzyme reaction enabled the detection of AFB1 at 0.2nM. We further lowered the detection limit of AFB1 to 0.01nM through chemiluminescence analysis by using chemiluminescence substrate of HRP. This assay enabled the detection of AFB1 in complex sample matrix, such as diluted white wine and maize flour. This assay provides a simple, sensitive and rapid method for AFB1 determination.

Eu3+ -labeled IgG-based time-resolved fluoroimmunoassay for highly sensitive detection of aflatoxin B1 in feed.

Aflatoxin B1 (AFB1 ) is a kind of toxic and carcinogenic mycotoxin. A time-resolved fluoroimmunoassay (TRFIA) was established for quantitative detection of AFB1 in feed using Eu3+ -labeled IgG as tracer. Monoclonal antibody (McAb) against AFB1 (9B11-D7) was prepared through immunization and cell fusion and was identified as high affinity, specificity and sensibility by enzyme-linked immunosorbent assay (ELISA). The 50% inhibition value (IC50 ) was 0.81 ng mL-1 , the limit of detection (LOD) was 0.10 ng mL-1 and detection range was 0.10-3.94 ng mL-1 . Goat anti-mouse immunoglobulin G (IgG) was modified by Eu3+ -DATT, generating Eu3+ -labeled IgG. Under optimal assay conditions, TRFIA was shown to be highly sensitive and specific in detection of AFB1 . The IC50 and LOD were 94.73 pg mL-1 and 3.55 pg mL-1 , respectively, and detection range was 3.55-1.11 × 103 pg mL-1 . Cross-reactivity with AFM1 , AFB2 , AFG1 and AFG2 was 31.26%, 37.6%, 127.46% and 35.74%, respectively, but zero with other analogues. In determination of AFB1 spiked in feed sample, TRFIA showed high accuracy and precision. The average recoveries ranged from 93.71% to 97.80%, and coefficient of variation was 1.25-3.73%. Good correlation between TRFIA and HPLC was demonstrated for determination of AFB1 in feeds, confirming the reliability of the developed method. The developed TRFIA exhibited good potential for employment in the ultrasensitive detection of AFB1 in feed and could be used to determine total aflatoxins. © 2017 Society of Chemical Industry.