Spiked Corn Samples Research Articles

Phage-displayed nanobody-based fluorescence-linked immunosorbent assay for the detection of Cry3Bb toxin in corn

Cry toxins have been widely used as biopesticides and applied in transgenic crops for the control of pests, raising public concerns for unforeseen environmental and health risks. In this study, five phage-displayed nanobodies against Cry3Bb toxin were selected from a naive nanobody library by screening. Using purified phage-displayed nanobody as capture antibody, a novel double-antibody sandwich fluorescence-linked immunosorbent assay (FLISA) based on quantum dot beads (QBs) was established for the determination of Cry3Bb toxin. The developed method exhibited a linear working range of 31.25–500 ng/mL and a low detection limit of 8.45 ng/mL. The recoveries of Cry3Bb toxin from spiked corn samples were ranging from 93.1% to 116.8% with a coefficient of variation lower than 9.4%. Molecular modeling results showed that key amino acids in the CDR3 region of nanobody P27 combine with Cry3Bb toxin through hydrogen bonds and hydrophobic interactions. These results indicated that the phage-displayed nanobody-based QBs-FLISA may provide a possible tool for detecting Cry3Bb toxin in corn.

Smartphone-based magneto-immunosensor on carbon black modified screen-printed electrodes for point-of-need detection of aflatoxin B1 in cereals

Considering the complexities and speed of modern food chains, there is an increasing demand for point-of-need detection of food contaminants, particularly highly regulated chemicals and carcinogens such as aflatoxin B1. We report a user-friendly smartphone-based magneto-immunosensor on carbon black modified electrodes for point-of-need detection of aflatoxin B1 in cereals. For buffered analyte solutions and a corn extract sample, the assay demonstrated a low limit of detection of 13 and 24 pg/mL, respectively. The assay was also highly reproducible, exhibiting mean relative standard deviations of 3.7% and 4.0% for the buffered analyte and corn extract samples. The applicability of the assay was validated on the basis of EU guidelines and the detection capability was lower than or equal to 2 μg/kg, which is the EU maximum residue limit for aflatoxin B1 in cereals. False-positive and false-negative rates were less than 5%. Additionally, an open-source android application, AflaESense, was designed to provide a simple interface that displays the result in a traffic-light-type format, thus minimizing user training and time for data analysis. AflaESense was used for smartphone-based screening of spiked corn samples containing aflatoxin B1 (0.1, 2, and 10 ng/mL), and naturally contaminated corn containing 0.15 ng aflatoxin B1/mL. The measured values were in close agreement with spiked concentrations (r2 = 0.99), with recovery values ranging between 80 and 120%. Finally, contaminated samples correctly triggered a red alert while the non-contaminated samples led to the display of a green color of AflaESense. To the best of our knowledge, this is the first smartphone-based electrochemical system effective for screening samples for contamination with aflatoxin B1.

Rapid and Label-Free Electrochemical Detection of Fumonisin-B1 Using Microfluidic Biosensing Platform Based on Ag-CeO2 Nanocomposite

Food contamination by mycotoxins is a major problem. Fumonisin-B1 (FU-B1) is one of the most abundantly found mycotoxins in products such as corn, rice, maize, and wheat, causing some of the most contagious diseases, such as pulmonary edema, hydrothorax, and immunosuppression, and is possibly carcinogenic. Its rapid and early detection in food products is therefore vital. We have fabricated an electrochemical microfluidic biosensing platform to detect FU-B1 with silver-ceria nanocomposite using maskless lithography. Structural characterizations confirmed nanocubes formation (CeO2) and spherical structures (Ag) with no impurities. The resulting nanobiochip showed an excellent linear response to antigen-FU-B1. linear detection was obtained from 10 pg ml−1−100 ng ml−1 with high sensitivity as 7.33 μA (log (ng. ml−1))−1cm−2. Limit of detection and limit of quantification were obtained as 1.5 pg. ml−1, and 3.9 pg. ml−1, respectively. Reynold’s number was found to be 0.79, showing the fluid’s laminar flow inside the channel. Interference studies showed specificity toward FU-B1 detection. Applicability in real food samples was assessed using spiked corn samples and showed recovery of up to 85% with excellent linear response. These results indicate that an electrochemical microfluidic nanobiochip can be used for rapid, label-free, and highly sensitive detection of antigen FU-B1.

An amplified electrochemical aptasensor for ochratoxin A based on DNAzyme-mediated DNA walker

• An amplified electrochemical aptasensor for ochratoxin A was developed. • Ag + -dependent DNAzyme was used to drive DNA walker for signal amplification. • The aptasensor showed higher sensitivity, lower LOD and good anti-interference. In this work, a novel signal-on electrochemical aptasensor was designed for ochratoxin A (OTA) detection based on DNAzyme-mediated DNA walker. Double-stranded DNA (dsDNA), formed by the hybridization between OTA aptamer and DNA walker, and Hairpin DNA (HP) containing adenosine ribonucleotide (rA) site were immobilized on the electrode surface. In the presence of OTA, the competitive binding of OTA with aptamer caused dsDNA to dissociate, resulting in that DNAzyme sequence in DNA walker was exposed. With the addition of Ag + , DNAzyme-mediated DNA walker was activated and sheared HP on the electrode surface cyclically, which reduced the negative charge density and improved the transfer rate on the electrode surface for signal amplification. The peak current increased by 4.58 times with addition of Ag + than that without addition of Ag + . The proposed aptasensor exhibited a good linear relationship in the OTA concentrations from 0.001 ng/mL to 5 ng/mL, and the limit of detection was 0.1 pg/mL. The aptasensor showed good anti-interference toward AFB 1 , FB 1 , and ZEN. The aptasensor was applied to detect OTA in spiked corn samples and wine samples, and the average recoveries were 94.5%–97.7% and 92.0%–97.8%, respectively.

A ratiometric electrochemical aptasensor for ochratoxin A detection

AbstractWith the superiority of providing a more accurate signal through their self‐referencing capability and reducing the environmental influence, a ratiometric electrochemical aptasensor was proposed for ochratoxin A (OTA) detection using methylene blue (MB) and ferrocene (Fc) as signal probes. The magnetic beads were used for the immobilization of OTA aptamer (Apt). When OTA and MB‐labeled complementary DNA (cDNA‐MB) were added, OTA combined with Apt, whereas cDNA‐MB released and existed in the supernatant after magnetic separation. By dropping the supernatant onto the electrode modified with HP‐Fc, the hairpin structure of HP‐Fc was opened by the hybridization between cDNA‐MB and HP‐Fc. As a result, MB was close to the electrode surface, whereas Fc was away from the electrode surface. Based on the value of IMB/IFc, OTA was detected sensitively and specifically. Under the optimal conditions, a linear relationship in the range of 0.005–50 ng/ml was obtained with the detection limit of 2.35 pg/ml. The aptasensor showed high anti‐interference toward AFB1, FB1, ZEN, and OTB. The aptasensor was applied to detect OTA in spiked corn samples, and the recoveries were 93–102%. This method provides a great potential for the development of sensitive biosensing platform for food safety.

Highly sensitive phage-magnetic-chemiluminescent enzyme immunoassay for determination of zearalenone

Here we demonstrate a novel phage-magnetic-chemiluminescent enzyme immunoassay (P-MCLEIA) for detection of zearalenone (ZEN). The P-MCLEIA was more efficient than conventional ELISA through several improvements. In the P-MCLEIA, magnetic nanoparticles were replaced of microplates as solid phases to reduce the whole incubation time within 40 min. Phage-mimotope was replaced of chemosynthetic antigen to improve the sensitivity of immunoassay. Chemiluminescence substrate was replaced of chromogenic substrate to further improve the sensitivity. The IC50 value of P-MCLEIA was 31.4 pg/mL, which was about 11 times lower than that of phage-magnetic-enzyme linked immunosorbent assay (P-MELISA) and 72 times lower than that of conventional ELISA. The LOD of P-MCLEIA was 4.3 pg/mL. Recovery study of P-MCLEIA was performed by analyzing ZEN levels in spiked corn samples, intra- and inter-assay recoveries were 80.0–119.8% and 82.7–112.7%, respectively. Furthermore, parallel analysis of natural corn samples showed a good correlation between the P-MCLEIA and high performance liquid chromatography.

Indirect Competitive Immunoassay on a Blu-ray Disc for Digitized Quantitation of Food Toxins.

We report herein a Blu-ray disc technology enabled immunoassay (namely, assay-on-a-Blu-ray) protocol for the quantitation of food toxins. In particular, commercial Blu-ray discs (BDs) are activated as substrates to create indirect competitive immunoassays with the aid of microfluidic channel plates for the quantitation of aflatoxins; an unmodified Blu-ray drive is employed to read the digitized signal (error counts generated from gold/silver-particle-enhanced binding sites); and a free disc-quality control software is adapted to process the raw data. The performance of this BD-based digital detection platform has been tested for the quantitation of aflatoxin B1 (AFB1) in spiked corn samples and validated with standard high-performance liquid chromatography measurements. The detection limit attained is as low as 0.27 ppb with a dynamic response range up to 200 ppb, which meets the standards established by government agencies worldwide for food products. We truly believe that the application potential of such a BD-technology-based, portable device for multiplex on-site quantitative analysis of food products as well as environmental and biomedical samples in real time is unlimited.

A fluorometric method for aptamer-based simultaneous determination of two kinds of the fusarium mycotoxins zearalenone and fumonisin B1 making use of gold nanorods and upconversion nanoparticles.

An aptamer-based assay for the determination of two different kinds of fusarium mycotoxins, i.e., zearalenone (ZEN) and fumonisin B1 (FB1), is presented. Based on theinner filter effect (IFE) strategy, the contents of ZEN and FB1 can be simultaneously quantified. It is making use of 65-nm gold nanorods (AuNRs), 20-nm upconversion nanoparticles (UCNPs), fluorescence dyes, and DNA sequences. In the absence of ZEN and FB1, theUCNPs and AuNRs associate through DNA sequences. Due to IFE effect, weak fluorescence signals are collected. In the presence of ZEN or FB1, UCNPs and AuNRs become unstable and partially separate from each other. This results in the recovery of fluorescence signals. Under 980-nm laser excitation, the logarithmic values of fluorescence signal intensities at 606nm and 753nm gradually increase with the concentration of ZEN and FB1 in the ranges 0.05-100μgL-1 (the coefficient of determination is 0.997) and 0.01-100ngL-1 (the coefficient of determination is 0.986), respectively. The limits of detection (LOD) of the fabricated assay for ZEN and FB1 are0.01μgL-1 and 0.003ngL-1, respectively. The proposed methodhas a high selectivity over other competitive mycotoxins, including aflatoxin B1, ochratoxin A, patulin andochratoxin B. The applicabilityof the assay was evaluated in the determination ofZEN and FB1 contents in spiked corn samples. The average recoveries ranged from 89.9 to 106.6%. This result confirms the practicality of this method. Graphical abstract Schematic representation of an aptamer-based fluorometric method for simultaneous determination of two kinds of the fusarium mycotoxins zearalenone and fumonisin B1.

Aptamer and gold nanorod-based fumonisin B1 assay using both fluorometry and SERS.

An aptamer-based assay is presented for the determination of fumonisin B1 (FB1). It is bimodal in that both surface-enhanced Raman spectroscopy (SERS) and fluorometry are applied for quantitation. It makes use of platinum-coated gold nanorod (AuNR) and DNA sequences. The complementary DNA of aptamer (cDNA) against FB1 is immobilized on the surface of AuNR. The aptamer of FB1 modified with Cy5.5 are complementarily hybridized with cDNA. In the absence of FB1, the aptamer and its cDNA associate. In this situation, strong SERS and weak fluorescence signals are obtained. In the presence of FB1, the aptamer disassociates with its cDNA and binds the target. As the concentration of FB1 increases, the SERS and fluorescence signal intensities of the mixture are gradually decreased and increased, respectively. Under optimized conditions, the SERS signal at 1366cm-1 decreases linearly in the 10-500pgmL-1 concentration range with the calibration equation of y = 1997lgx-594 (the coefficient of determination is 0.998). The fluorescence signal at 670nm increases linearly in the 10-250pgmL-1 concentration range with the calibration equation of y = 500lgx-383 (the coefficient of determination is 0.991). The assay was applied to the determination of FB1 contents in spiked corn samples. The average recoveries ranged from 92 to 107%, confirming the practicality of this method. The results obtained by this assay are in good agreement with that of LC-MS/MS method. Graphical abstractSchematic illustration of a bimodal aptasensor based on surface enhanced Raman scattering (SERS) and fluorescence change for the detection of fumonisin B1 (FB1).

MIL-101(Cr)/MWCNTs-functionalized melamine sponges for solid-phase extraction of triazines from corn samples, and their subsequent determination by HPLC-MS/MS

In the presented work, we proposed the use of polyvinylidene difluoride (PVDF) as a coupling agent to load MIL-101(Cr) and carboxylated multi-walled carbon nanotubes (MWCNTs) on the skeleton of melamine sponge (MeS) for the preparation of novel extraction media with high-efficiency. The morphology and structure of this composite were characterized by scanning electron microscopy (SEM) analyses. The obtained MIL-101(Cr)/MWCNTs functionalized MeS(MIL/M-MeS) cube was used as a sorbent for the solid-phase extraction (SPE) of six triazines (Atraton, Simetryne, Prometon, Ametryn, Propazine, and Prometryn) spiked corn samples. Several parameters that may affect the extraction efficiencies, including type and volume of extraction solvent, times of ultrasonic extraction, adsorption, and desorption; type and volume of desorption solvent, were optimized. This was followed by elution containing the herbicides, which were quantified using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Under optimum conditions, the analytical method for the preparation of MIL/M-MeS cube produced satisfactory recoveries and repeatability. The LODs of the method when applied to spiked corn samples were in the range of 0.01–0.04 ng g−1 (LODs = 3δ/k). The relative intra- and inter-day recoveries ranged from 90.30 to 116.50% and 92.38–116.24%, respectively, and relative standard deviations ranged from 1.08 to 12.32% and 5.36–16.23%, respectively.

The determination of Ochratoxin A based on the electrochemical aptasensor by carbon aerogels and methylene blue assisted signal amplification

In this work, a novel aptamer-based electrochemical biosensor was developed for the determination of Ochratoxin A (OTA) by using carbon aerogels (CAs) and methylene blue (MB) as signal amplification strategy. CAs was used as carrier to load the abundant of complementary DNA (cDNA), which could enhance the hybridization between CAs-cDNA and aptamer immobilized on the electrode surface, thus provide more double-stranded DNA for MB intercalation. The current of MB on the CAs-cDNA/apt/AuE sensor was twice that on the cDNA/apt/AuE sensor, which indicated that the CAs with high surface area enabled a higher loading of the cDNA and absorbed more MB, thus realized the signal amplification strategy. The optimum experimental conditions including MB incubation time of 15 min, aptamer concentration of 4.0 μmol/L, hybridization time of 2.0 h, and OTA incubation time of 18 min were obtained. The change of peak current was linearly proportional to the OTA concentration in the range of 0.10–10 ng/mL with the actual detection limit of 1.0 × 10−4 ng/mL. The experimental results showed that the prepared CAs-cDNA/apt/AuE exhibited good specificity, acceptable reproducibility and repeatability. This sensor was applied to detect OTA in the spiked corn samples, and obtained an acceptable average recovery of 89%.

Label-Free Impedance Sensing of Aflatoxin B₁ with Polyaniline Nanofibers/Au Nanoparticle Electrode Array.

Aflatoxin B1 (AFB1) is produced by the Aspergillus flavus and Aspergillus parasiticus group of fungi which is most hepatotoxic and hepatocarcinogenic and occurs as a contaminant in a variety of foods. AFB1 is mutagenic, teratogenic, and causes immunosuppression in animals and is mostly found in peanuts, corn, and food grains. Therefore, novel methodologies of sensitive and expedient strategy are often required to detect mycotoxins at the lowest level. Herein, we report an electrochemical impedance sensor that selectively detects AFB1 at the lowest level by utilizing polyaniline nanofibers (PANI) coated with gold (Au) nanoparticles composite based indium tin oxide (ITO) disk electrodes. The Au-PANI nanocomposites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) spectroscopy, and electrochemical impedance spectroscopy (EIS). The composite electrode exhibited a 14-fold decrement in |Z|1 Hz in comparison with the bare electrode. The Au-PANI acted as an effective sensing platform having high surface area, electrochemical conductivity, and biocompatibility which enabled greater loading deposits of capture antibodies. As a result, the presence of AFB1 was screened with high sensitivity and stability by monitoring the changes in impedance magnitude (|Z|) in the presence of a standard iron probe which was target specific and proportional to logarithmic AFB1 concentrations (CAFB1). The sensor exhibits a linear range 0.1 to 100 ng/mL with a detection limit (3σ) of 0.05 ng/mL and possesses good reproducibility and high selectivity against another fungal mycotoxin, Ochratoxin A (OTA). With regard to the practicability, the proposed sensor was successfully applied to spiked corn samples and proved excellent potential for AFB1 detection and development of point-of-care (POC) disease sensing applications.

A colorimetric immunoassay based on glucose oxidase-induced AuNP aggregation for the detection of fumonisin B1

Naked-eye readout colorimetric signal-based assays are promising for high-throughput screening detection and point-of-care diagnostics in resource-constrained regions. Here, a novel direct competitive plasmonic enzyme-linked immunosorbent assay (dc-pELISA) based on gold nanoparticle (AuNP) aggregation with highly sensitive and robust naked-eye readout signal was developed and used to detect fumonisin B1 (FB1). AuNP aggregation was induced by a horseradish peroxidase (HRP)/hydrogen peroxide (H2O2)/tyramine (TYR) system, resulting in a dramatic color change from red to blue. In this system, H2O2 was produced via GOx-mediated glucose oxidation reaction, and FB1–GOx was used as a competitive antigen. The proposed pELISA demonstrated good linear detection of FB1 from 3.125 ng mL−1 to 25 ng mL−1 with a vivid color change from deep blue to red and a cutoff limit of 12.5 ng mL−1 observed by the naked eye. The average recoveries for FB1-spiked corn samples ranged from 76.5% to 96.8% with a relative standard derivation of 4.88~ 16.4%. Meanwhile, the proposed method exhibited excellent agreement (R2 = 0.927) with the conventional ELISA method in blindly detecting FB1 spiked corn samples. Additionally, the results of the proposed method for FB1 positive corn samples also showed a high consistency with those of the ultra performance liquid chromatography method. These results indicated acceptable accuracy and precision of the proposed colorimetric ELISA for quantitative detection of FB1 in actual corn samples.

Magnetically controlled fluorescence aptasensor for simultaneous determination of ochratoxin A and aflatoxin B1

Development of an efficient method for the simultaneous detection of two highly concerning mycotoxins, ochratoxin A (OTA) and aflatoxin B1 (AFB1), is of great significance on food safety monitoring. Herein, a magnetically controlled fluorescence aptasensor for simultaneous determination of OTA and AFB1 has been successfully developed. The working principle of the aptasensor is based on the specific aptamer-mycotoxin recognition and further leads to the partial release of two distinguishable fluorescence labels from the magnetic carriers. Through the magnetic separation, the reporter probes in the supernatant solution can be collected and converted into a sensitive fluorescence signal with dual emission peaks. This aptasensor provided a wide detection range of 2 pg mL−1 − 5 ng mL−1 for OTA and 5 pg mL−1 − 10 ng mL−1 for AFB1. The new easy-to-wash and simple-to-use approach offers a simultaneous and high selective detection with high sensitivity (limits of detection of 0.67 and 1.70 pg mL−1 for OTA and AFB1, respectively). Remarkable accuracy (relative standard deviation < 5.6%) during the mycotoxins determination as well as excellent quantitative recoveries (95–108%) during the analysis of the spiked corn samples were also achieved. This simple aptasensing scheme provides a new avenue for high throughput screen of dual mycotoxins due to its simple manipulation, short assay times, high selectivity and sensitivity.

A Lateral Flow Strip Based Aptasensor for Detection of Ochratoxin A in Corn Samples.

Ochratoxin A (OTA) is a mycotoxin identified as a contaminant in grains and wine throughout the world, and convenient, rapid and sensitive detection methods for OTA have been a long-felt need for food safety monitoring. Herein, we presented a new competitive format based lateral flow strip fluorescent aptasensor for one-step determination of OTA in corn samples. Briefly, biotin-cDNA was immobilized on the surface of a nitrocellulose filter on the test line. Without OTA, Cy5-labeled aptamer combined with complementary strands formed a stable double helix. In the presence of OTA, however, the Cy5-aptamer/OTA complexes were generated, and therefore less free aptamer was captured in the test zone, leading to an obvious decrease in fluorescent signals on the test line. The test strip showed an excellent linear relationship in the range from 1 ng·mL−1 to 1000 ng·mL−1 with the LOD of 0.40 ng·mL−1, IC15 value of 3.46 ng·mL−1 and recoveries from 96.4% to 104.67% in spiked corn samples. Thus, the strip sensor developed in this study is an acceptable alternative for rapid detection of the OTA level in grain samples.

Development of a rapid magnetic bead-based immunoassay for sensitive detection of zearalenone

Here we demonstrate a novel magnetic bead-based enzyme-linked immunosorbent assay (MB-ELISA) for zearalenone (ZEN) detection. Firstly, an anti-ZEN monoclonal antibody (mAb) was prepared by hybridoma technique, and immobilized on carboxyl modified MBs to obtain mAb-MBs. In addition, the biotinylated ZEN-BSA was labelled by streptavidin-HRP for use as competitor. Based on the mAb-MBs and streptavidin-HRP labelled ZEN-BSA, a MB-ELISA which contains only one 20 min antigen-antibody reaction step and takes no more than 45 min for dozens of samples analysis was developed. The half maximal inhibitory concentration (IC50) and limit of detection (LOD) of the MB-ELISA are 1.78 ng/mL and 0.13 ng/mL, respectively. And the MB-ELISA working range for corn samples analysis is from 5.0 μg/kg to 255.2 μg/kg. The recoveries for ZEN spiked corn samples ranged from 82.3 to 110.5% with coefficient of variation (CV) under 8.9%. For natural corn samples analysis, the results of MB-ELISA showed good agreement with the results of conventional direct competitive ELISA (R2 = 0.9742).

Immuno Affinity SELEX for Simple, Rapid, and Cost-Effective Aptamer Enrichment and Identification against Aflatoxin B1.

Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower Kd values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ – 53.74 ng) and 78.3 to 94.22% (LOQ – 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification.

An Electrochemical Immunosensor for Rapid and Sensitive Detection of Mycotoxins Fumonisin B1 and Deoxynivalenol

We report an electrochemical immunosensing method for rapid and sensitive detection of two mycotoxins, fumonisin B1 (FB1) and deoxynivalenol (DON). A disposable screen-printed carbon electrode (SPE) was used as sensing platform. The working electrode part of SPE was modified by gold nanoparticles (AuNPs) and polypyrrole (PPy)-electrochemically reduced graphene oxide (ErGO) nanocomposite film for effective anti-toxin antibody immobilization, enhanced electrical conductivity, and biocompatibility. Under optimized test conditions, the limit of detection and linear range achieved for FB1 were 4.2ppb and 0.2 to 4.5ppm (%RSD=4.9%); and the corresponding values for DON were 8.6ppb and 0.05 to 1ppm (%RSD=5.7%). The immunosensor can specifically detect the target toxin in co-existing toxins environment. The sensor exhibited high sensitivity and low matrix interference when tested using extracts obtained from spiked corn samples. Hence, our electrochemical immunosensing scheme can be adopted for highly sensitive and rapid detection of multiple co-contaminant mycotoxins in food and feed products.

Determination of Citrinin Using Molecularly Imprinted Solid Phase Extraction Purification, HPLC Separation, and Fluorescence Detection

A liquid chromatography based method to detect citrinin in corn was developed using molecularly imprinted solid phase extraction (MISPE) sample clean-up. Molecularly imprinted polymers were synthesized using 1,4-dihydroxy-2-naphthoic acid as the template and an amine functional monomer. Density functional calculations suggest the mimic template interacts with the functional monomer in a similar manner as citrinin. Freundlich isotherm analysis indicated the template provided a significant imprinting effect for citrinin binding. A high performance liquid chromatography with fluorescence detection (HPLC-FD) method to detect citrinin in maize was developed utilizing the imprinted polymers for sample clean-up (excitation at 330 nm; emission at 500 nm). Recoveries of citrinin in spiked corn samples (0.03–3 µg g−1) were between 82.3–91.5%. This study demonstrates that molecularly imprinted polymers are applicable in the solid phase extraction clean-up of corn samples for citrinin determination by HPLC-FD.

A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples.

A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was developed for rapid and sensitive detection of zearalenone (ZEN). The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = −0.4287x + 0.3132 (R2 = 0.9904). The working range was 0.07–2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%–111.9% and 91.7%–114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R2 = 0.9283). We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories.