Ochratoxin A Binding Research Articles

Top-Down Computational Design of Molecule Recognition Peptides (MRPs) for Enzyme-Peptide Self-Assembly and Chemiluminescent Biosensing.

The recognition of small molecules plays a crucial role in disease diagnosis, environmental assessment, and food safety. Currently, their recognition elements predominantly rely on antibodies and aptamers while suffering from a limitation of the complex screening process due to the low immunogenicity of small molecules. Herein, we present a top-down computational design strategy for molecule recognition peptides (MRPs) for enzyme-peptide self-assembly and chemiluminescence biosensing. Taking ochratoxin A (OTA) as an illustrative example, human serum albumin (HSA) was selected as the parental protein due to its high affinity for OTA binding. Through iterative computational simulations involving the binding domain of the HSA-OTA complex, our strategy identified a specific 15-mer MRP (RLKCASLKFGERAFK), which possesses excellent binding affinity (38.02 ± 1.24 nM) against OTA. Molecular dynamics simulations revealed that the 15-mer MRP unfolds into a flexible short chain with high affinity for OTA, but exhibits weak or no binding affinity with five structurally similar mycotoxins. Furthermore, we developed a novel enzyme-peptide self-assembly approach mediated by calcium(II) to obtain nanoflowers, which integrates both the recognition element (MRP) and the signal translator (enzyme) for chemiluminescence biosensing. The assembled nanoflowers allow MRPs to be directly utilized as a tracer for OTA biosensing without labeling or secondary antibodies. This computational-to-application approach offers a new route for small-molecule recognition.

Chlorophyll-Amended Organoclays for the Detoxification of Ochratoxin A.

Climate change has been associated with outbreaks of mycotoxicosis following periods of drought, enhanced fungal growth, and increased exposure to mycotoxins. For detoxification, the inclusion of clay-based materials in food and drinking water has resulted in a very promising strategy to reduce mycotoxin exposure. In this strategy, mycotoxins are tightly sorbed to high-affinity clay particles in the gastrointestinal tract, thus decreasing bioavailability, uptake to blood, and potential toxicity. This study investigated the ability of chlorophyll and chlorophyllin-amended montmorillonite clays to decrease the toxicity of ochratoxin A (OTA). The sorption mechanisms of OTA binding to surfaces of sorbents, as well as binding parameters such as capacity, affinity, enthalpy, and free energy, were examined. Chlorophyll-amended organoclay (CMCH) demonstrated the highest binding (72%) and was better than the chlorophyllin-amended hydrophilic clay (59%), possibly due to the hydrophobicity of OTA (LogP 4.7). In silico studies using molecular dynamics simulations showed that CMCH improves OTA binding in comparison to parent clay in line with experiments. Simulations depicted that chlorophyll amendments on clay facilitated OTA molecules binding both directly, through enhancing OTA binding on the clay, or predominantly indirectly, through OTA molecules interacting with bound chlorophyll amendments. Simulations uncovered the key role of calcium ions in OTA binding, particularly in neutral conditions, and demonstrated that CMCH binding to OTA is enhanced under both neutral and acidic conditions. Furthermore, the protection of various sorbents against OTA-induced toxicity was carried out using two living organisms (Hydra vulgaris and Caenorhabditis elegans) which are susceptible to OTA toxicity. This study showed the significant detoxification of OTA (33% to 100%) by inclusion of sorbents. Organoclay (CMCH) at 0.5% offered complete protection. These findings suggest that the chlorophyll-amended organoclays described in this study could be included in food and feed as OTA binders and as potential filter materials for water and beverages to protect against OTA contaminants during outbreaks and emergencies.

Study of dual binding specificity of aptamer to ochratoxin A and norfloxacin and the development of fluorescent aptasensor in milk detection

Target specificity, one of aptamer characteristics that determine recognition efficiency of biosensors, is generally considered to be an intrinsic property of aptamer. However, a high-affinity aptamer may have additional target binding specificity, little is known about the specificity of aptamer binding to multiple targets, which may result in false-positive results that hinder the accuracy of detection. Herein, an aptamer OBA3 with dual target ochratoxin A (OTA) and norfloxacin (NOR) was used as an example to explore the binding specificity mechanism and developed rapid fluorescent aptasensing methods. The nucleotide 15th T of aptamer OBA3 was demonstrated to be critical for specificity and affinity binding of target OTA via site-saturation mutagenesis. Substituting the 15th T base for C base could directly improve recognition specificity of aptamer for NOR and remove the binding affinity for OTA. The combination of π-π stacking interactions, salt bridges and hydrogen bonds between loop pocket of aptamer and quinolone skeleton, piperazinyl group may contributes to the fluoroquinolone antibiotics (NOR and difloxacin)-aptamer recognition interaction. Based on this understanding, a dual-aptamer fluorescent biosensor was fabricated for simultaneous detection of OTA and NOR, which has a linear detection range of 50–6000 nM with a detection limit of 31 nM for OTA and NOR. Combined with T15C biosensor for eliminating interference of OTA, the assay was applied to milk samples with satisfactory recovery (94.06–100.93%), which can achieve detection of OTA and NOR individually within 40 min.

Dual-mode aptasensor based on a coumarin–benzothiazole fluorescent and colorimetric probe for label-free and visual detection of ochratoxin A

Ochratoxin A (OTA) is regarded as a harmful mycotoxin that severely influences human health by increasing the risk of diseases. Although conventional quantitative methods for OTA detection have been developed, there remain drawbacks of limited signal transduction, bulky instrument requirements, high cost, and sophisticated sample pretreatment. Herein, we successfully synthesize a coumarin-benzothiazole molecule named DR1 to construct a dual-mode aptasensor (DR1/OBA) by simultaneously producing fluorescent and colorimetric signals when binding to OTA binding aptamer (OBA). In fluorescent mode, the fluorescence of DR1/OBA will be switched off and provide rapid, label-free, and sensitive detection efficacies via the OTA competition. In colorimetric mode, the purple-color of DR1 tends to a blue-color state with a red-shifted absorbance by forming DR1/OBA. The blue-shifted absorbance will be observed with OTA competition and the purple-color state of DR1 can be recovered. Based on this unique phenomenon, a smartphone-based detection platform has been established to convert the color change into RGB values and realize the visual detection for spiked OTA quantification in real samples. As expected, the dual-mode aptasensor allows portable, reliable, sensitive, and visual detection for OTA in sophisticated scenarios. It will hold great potential in advancing research in mycotoxins and the development of aptamer-based biosensing.

Structure-switching aptasensors for sensitive detection of ochratoxin A.

Ochratoxin A (OTA) is a toxic metabolite commonly found in various foods and feedstuffs. Accurate and sensitive detection of OTA is needed for food safety and human health. Based on a common OTA-binding aptamer (OTABA), two structure-switching OTABAs, namely OTABA4 and OTABA3, were designed by configuring a split G-quadruplex and a split G-triplex, respectively, at the two ends of OTABA to construct aptasensors for the detection of OTA. The OTABA, G-quadruplex, and G-triplex all can capture the thioflavin T (ThT) probe, thereby enhancing the fluorescence intensity of ThT. Bonding with OTA could change the conformations of OTABA and G-quadruplex or G-triplex regions, resulting in the release of the captured ThT and diminution of its fluorescence intensity. Dual conformation changes in structure-switching OTABA synergistically amplified the fluorescence signal and improved the sensitivity of the aptasensor, especially for that with OTABA3. The detection limits of the OTABA4-ThT and OTABA3-ThT systems for OTA were 0.28 and 0.059 ng ml-1 , with a 1.4-fold and 6.7-fold higher sensitivity than that of the original OTABA-ThT system, respectively. They performed well in corn and peanut samples and met the requirements of the food safety inspections.

A label-free colorimetric 3D paper-based device for ochratoxin A detection using G-quadruplex/hemin DNAzyme with a smartphone readout

The colorimetric sensor usually depends on enzyme-mediated signal amplification to achieve trace analysis of ochratoxin A (OTA) residues in food samples. However, the enzyme labeling and manual addition of reagents steps increased assay time and operation complexity, restricting their application in point-of-care testing (POCT). Herein, we report a label-free colorimetric device integrating a 3D paper-based analytical device and a smartphone as handheld readout for rapid and sensitive detection of OTA. Using vertical-flow design, the paper-based analytical device enables the specific recognition of target and self-assembly of G-quadruplex (G4)/hemin DNAzyme to be performed, then employs DNAzyme for transducing the OTA binding event signal into a colorimetric signal. The design of independent functional units, including biorecognition unit, self-assembly unit and colorimetric units, which can address crowding and disorder of biosensing interfaces and improve the recognition efficiency of aptamer (apta). In addition, we eliminated signal losses and nonuniform coloring by introducing carboxymethyl chitosan (CMCS) to obtain perfectly focused signals on colorimetric unit. On the basis of parameter optimization, the device exhibited a detection range of 0.1–500 ng/mL and a detection limit of 41.9 pg/mL for OTA. Importantly, good results were obtained in spiked real samples, indicating applicability and reliability of developed device.

Sensitive microscale thermophoresis assay for rapid ochratoxin A detection with fluorescently labeled engineered aptamer.

Ochratoxin A (OTA) is a widespread mycotoxin that causes contamination in a variety of foodstuffs and environments, inducing great health risks to humans and animals. Rapid and sensitive detection of OTA is necessary for food safety, environmental health, and risk assessment. Herein, we report an aptamer microscale thermophoresis (MST) assay for OTA, with the unique merits of ratiometric analysis, rapid measurement, simple operation, high sensitivity, low sample consumption, and high throughput. A fluorescein (FAM)-labeled high-affinity DNA aptamer with a G-quadruplex and duplex structure was used as the recognition element for OTA, and MST, which measures the fluorescence responses of the sample solution inside capillaries to a mild temperature increase generated by infrared laser heating, was employed for signal generation. Upon OTA binding, the FAM-labeled aptamer probe underwent changes in conformation and stability, and the bound and unbound aptamer probes showed significant differences in their MST signals. To achieve sensitive detection of OTA with a large signal change, we systematically characterized aptamers with different stem lengths, which had large effects on the MST responses of the aptamer probes to OTA. We found that a 32-mer aptamer with FAM label at the 3' end gave a sensitive MST response to OTA, allowing OTA detection within seconds with a detection limit of 0.98 nM under optimal experimental conditions. This aptamer MST assay shows potential in real sample analysis and broad applications.

Surface Functionalization Strategies of Polystyrene for the Development Peptide-Based Toxin Recognition.

The development of a robust surface functionalization method is indispensable in controlling the efficiency, sensitivity, and stability of a detection system. Polystyrene (PS) has been used as a support material in various biomedical fields. Here, we report various strategies of polystyrene surface functionalization using siloxane derivative, divinyl sulfone, cyanogen bromide, and carbonyl diimidazole for the immobilization of biological recognition elements (peptide developed to detect ochratoxin A) for a binding assay with ochratoxin A (OTA). Our objective is to develop future detection systems that would use polystyrene cuvettes such as immobilization support of biological recognition elements. The goal of this article is to demonstrate the proof of concept of this immobilization support. The results obtained reveal the successful modification of polystyrene surfaces with the coupling agents. Furthermore, the immobilization of biological recognition elements, for the OTA binding assay with horseradish peroxidase conjugated to ochratoxin A (OTA-HRP) also confirms that the characteristics of the functionalized peptide immobilized on polystyrene retains its ability to bind to its ligand. The presented strategies on the functionalization of polystyrene surfaces will offer alternatives to the possibilities of immobilizing biomolecules with excellent order- forming monolayers, due to their robust surface chemistries and validate a proof of concept for the development of highly efficient, sensitive, and stable future biosensors for food or water pollution monitoring.

Aptamer Molecular Beacon Sensor for Rapid and Sensitive Detection of Ochratoxin A

Ochratoxin A (OTA) is a carcinogenic fungal secondary metabolite which causes wide contamination in a variety of food stuffs and environments and has a high risk to human health. Developing a rapid and sensitive method for OTA detection is highly demanded in food safety, environment monitoring, and quality control. Here, we report a simple molecular aptamer beacon (MAB) sensor for rapid OTA detection. The anti-OTA aptamer has a fluorescein (FAM) labeled at the 5' end and a black hole quencher (BHQ1) labeled at the 3' end. The specific binding of OTA induced a conformational transition of the aptamer from a random coil to a duplex-quadruplex structure, which brought FAM and BHQ1 into spatial proximity causing fluorescence quenching. Under the optimized conditions, this aptamer sensor enabled OTA detection in a wide dynamic concentration range from 3.9 nM to 500 nM, and the detection limit was about 3.9 nM OTA. This method was selective for OTA detection and allowed to detect OTA spiked in diluted liquor and corn flour extraction samples, showing the capability for OTA analysis in practical applications.

An Alkyne-Mediated SERS Aptasensor for Anti-Interference Ochratoxin A Detection in Real Samples.

Avoiding interference and realizing the precise detection of mycotoxins in complex food samples is still an urgent problem for surface-enhanced Raman spectroscopy (SERS) analysis technology. Herein, a highly sensitive and specific aptasensor was developed for the anti-interference detection of Ochratoxin A (OTA). In this aptasensor, 4-[(Trimethylsilyl) ethynyl] aniline was employed as an anti-interference Raman reporter to prove a sharp Raman peak (1998 cm-1) in silent region, which could avoid the interference of food bio-molecules in 600-1800 cm-1. 4-TEAE and OTA-aptamer were assembled on Au NPs to serve as anti-interference SERS probes. Meanwhile, Fe3O4 NPs, linked with complementary aptamer (cApts), were applied as capture probes. The specific binding of OTA to aptamer hindered the complementary binding of aptamer and cApt, which inhibited the binding of SERS probes and capture probes. Hence, the Raman responses at 1998 cm-1 were negatively correlated with the OTA level. Under the optimum condition, the aptasensor presented a linear response for OTA detection in the range of 0.1-40 nM, with low detection limits of 30 pM. In addition, the aptasensor was successfully applied to quantify OTA in soybean, grape and milk samples. Accordingly, this anti-interference aptasensor could perform specific, sensitive and precise detection of OTA in real samples, and proved a reliable reference strategy for other small-molecules detection in food samples.

Development of a label-free electrochemical aptasensor for ultrasensitive detection of ochratoxin A

The prevalent ochratoxin A (OTA) contamination in food has increased the urgency to develop sensitive and accurate detection methods. This study reported a conveniently fabricated label-free electrochemical (EC) aptasensor for ultrasensitive detection of OTA based on the competitive binding of OTA with its specific aptamer without any signal amplification strategy. A gold electrode (AuE) was modified with a compact layer of double-stranded DNA (dsDNA), obtained via OTA aptamer and complementary DNA (cDNA) hybridization, to act as a barrier for redox probe [Fe(CN) 6 ] 3-/4- to AuE access. This led to a high initial electron-transfer resistance (R ct ), recorded via electrochemical impedance spectroscopy (EIS). Since the aptamer displayed a higher affinity to OTA than cDNA, the added OTA triggered aptamer dissociation from AuE to form the OTA-aptamer complex, obviously decreasing the R ct . The target-induced variation in the resistance response strongly depended on the OTA concentration. Optimum conditions improved the linear relationship between R ct and the logarithm of OTA concentration in a range of 0.05–10 ng/mL with a limit of detection (LOD) of 0.05 ng/mL for OTA. Recoveries of 74.8–105% with relative standard deviations (RSDs) of 8.86–13.09% were obtained in the spiked malt samples. The Real-world detection of OTA in the actual malt samples confirmed the reliability of the convenient, cost-effective, and highly specific impedimetric aptasensor, exhibiting significant promise for extended application in food control to identify more hazardous contaminants by altering the corresponding aptamers. • A label-free electrochemical impedimetric aptasensor for OTA detection was developed. • No extra coupling medium was involved for immobilizing the aptamers. • The aptasensor achieved high sensitivity for OTA without signal amplification. • The impedimetric aptasensor succeeded in real malt samples with satisfactory recoveries. • The aptasensor can be extended to more contaminants in foods via replacing the corresponding aptamers.

Determining the adsorption capacity and stability of Aflatoxin B1, Ochratoxin A, and Zearalenon on single and co-culture L. acidophilus and L. rhamnosus surfaces

This study assessed the adsorption capacity and stability of Aflatoxin B1 (AFB1), Ochratoxin A (OTA), and Zearalenon (ZEA) via Lactobacillus acidophilus ATCC 4356 and Lactobacillus rhamnosus ATCC 7469 individually and in combination with each other. Detoxifying efficiency depends on lactic acid bacteria species (hydrophobicity of cell wall), pH, temperature, time and mycotoxin type. According to the genetic algorithm results, the highest adsorption capacity (qe) of toxin for L. acidophilus + L. rhamnosus after washing with ethanol and chloroform decreased to (8.487 and 10.309 μg kg−1; AFB1), (9.464 and 9.014 μg kg−1; OTA), and (162.020 and 144.047 μg kg−1; ZEA), which is significantly less than before washing (9.872, 9.834, and 180.993) for AFB1, OTA, and ZEA after 24 h incubation at 37 °C. The surface hydrophobicity in turn toxin binding stability and capacity of the strains increased by increasing the incubation temperature from 27° to 37°C after 24 h. The affinity of AFB1 and OTA toxins to surface of LAB showed the most and the least content. For investigation of the stability binding of the AFB1, OTA and ZEA on LAB surfaces, toxin+LAB complexes were washed with solvents of different polarities. The bacteria–toxin complex stability showed the trend AFB1 > OTA> ZEA for toxin release in ethanol and ZEA> OTA> AFB1 for chloroform based on the difference in their polarity. Practical applicationIn the present study, the investigation of the efficiency of AFB1, OTA and ZEA adsorption on single and co-culture L. acidophilus and L. rhamnosus, on the basis of bacterial cell wall hydrophobicity and toxin polarity was studied. Food composites can have polarity differences that affect the stability of the toxin-bacteria interaction. Therefore, the monitoring of the LAB + mycotoxins capacity and stability through washing with solvents of different polarities, modeling the AFB1, OTA and ZEA binding capacity and stability before and after washing at different dough proofing times and temperatures has been investigated.

Surface-enhanced Raman spectroscopy aptasensor for simultaneous determination of ochratoxin A and zearalenone using Au@Ag core-shell nanoparticles and gold nanorods.

Thedesign and fabrication of a surface-enhanced Raman scattering (SERS) aptasensor forsimultaneous detection of zearalenone (ZEN) and ochratoxin A (OTA) in wheat and corn samplesis described. The capture and reporter probes were SH-cDNA-modified gold nanorods and SH-Apt-modified Au@Ag core-shell nanoparticles, respectively. After recognizing OTA and ZEN aptamers and complementary strands (SH-cDNA), the reporter probe generated a strong SERS signal. The preferred binding of OTA and ZEN aptamers to OTA and ZEN, respectively, caused reporter probes to release the capture probes, resulting in a linear decrease in SERS intensity. The detection of OTA showed good linearity with an R2 value of 0.986, which could be maintained across a wide concentration range (0.01 to 100 ng/mL), with the limit of detection of 0.018 ng/mL. Fordetection of ZEN, good linearity with an R2 value of 0.987 could be maintained across a wide concentration range (0.05 to 500 ng/mL), with 0.054 ng/mL as the limit of detection. Goodaccuracy (relative standard deviation < 4.2%) during mycotoxin determination as well as excellent quantitative recoveries (96.0-110.7%) during the analysis of spiked real samples was achieved. The proposed SERS aptasensor exhibited excellent performance in the detection of OTA and ZEN in real food samples. Hence, by simply changing the aptamer, this new model can be applied tothe detection of multiple mycotoxins in the food industry.

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.

Cascade strand displacement reaction-assisted aptamer-based highly sensitive detection of ochratoxin A

Ochratoxin A (OTA) is a toxic metabolite that is widely distributed in food products. Herein, we proposed a new fluorescent aptasensor for OTA detection by using cascade strand displacement reaction. The binding of OTA and OTA aptamer on magnetic beads surface inhibited its hybridization with complementary DNA, and subsequently initiated the strand displacement reaction that induced amplified fluorescence signal. By tracing fluorescence response, our method demonstrated an improved detection limit of 0.63 ng/mL, a short assay time of 110 min, and a satisfactory detection specificity by using ochratoxin B, aflatoxin B1, and zearalenone as control toxins. Recovery studies were conducted by spiking OTA in real food samples, including white wine, red wine, cereal drink, coffee beverage and tea beverage, and confirmed desirable accuracy and practical applicability of our method. Therefore, our method may have a great potential use in the food quality control in the future.

Specific Coordination between Zr-MOF and Phosphate-Terminated DNA Coupled with Strand Displacement for the Construction of Reusable and Ultrasensitive Aptasensor.

Electrochemical aptasensors involved in chemical labeling are often single-use and sensitivity-limited because the probes are commonly single-point labeled and irreversible. In this work, the specific coordination between Zr4+ and phosphate group (-PO43-) was employed to construct a new aptasensor that is highly sensitive and reusable, using Ochratoxin A (OTA) as the test model. The OTA binding aptamer (OBA) was hybridized with the thiolated supporting sequence (TSS) immobilized on the surface of a gold electrode. The UiO-66 with a formula of [Zr6O4(OH)4(BDC)6], one of the class of Zr metal-organic frameworks (MOFs), was then particularly grafted on the terminal of OBA through the specific coordination between Zr4+ and 5'-PO43-, i.e., the Zr-O-P coordination bond. Similarly, as much as the 5'-PO43- and 3'-methylene blue dual-labeled sequences (DLS) were further assembled on UiO-66 due to the large surface area of MOF and rich active sites of Zr4+. Owing to the specific coordination for signal amplification, the developed aptasensor shows greatly enhanced sensitivity. A wide detection range from 0.1 fM to 2.0 μM and an ultralow detection limit of 0.079 fM (S/N = 3) for OTA were obtained. Additionally, the TSS can rehybridize with a new OBA to regenerate the aptasensor but without complicated pretreatments, enabling a aptasensor that is readily reusable for OTA detection. The aptasensor was successfully applied for OTA detection in the red wine samples, demonstrating a promising prospect for food safety monitoring.

Intrinsic “Turn-On” Aptasensor Detection of Ochratoxin A Using Energy-Transfer Fluorescence

Ochratoxin A (OTA) is an intrinsically fluorescent phenolic mycotoxin that contaminates a wide range of food products and is a serious health threat to animals and humans. An OTA binding aptamer (OTABA) that folds into an antiparallel G-quadruplex (GQ) in the absence and presence of target OTA has been incorporated into a vast variety of aptasensor platforms for OTA detection. The development of a simple, aptamer-based approach would allow for detection of the toxin without the use of complex analytical instrumentation, which has been the gold standard for OTA detection thus far. However, to date none of the aptasensor platforms have utilized the natural fluorescence of the phenolic toxin for detection. Herein, we report that OTA binding to OTABA involves pi-stacking interactions that leads to GQ-to-toxin energy transfer (ET), which affords a 'turn-on' fluorescence self-signaling platform in which the emission of the aptamer‒target complex is enhanced in comparison to the free toxin alone. Selective excitation of the GQ‒OTA complex at 256 nm leads to a 4-fold enhancement in OTA fluorescence. The GQ‒OTA ET detection platform boasts a limit of detection ~ 2 ng/mL, which is comparable to a previously demonstrated FRET immunoassay platform for OTA detection, and displays excellent OTA selectivity and recovery from red wine samples.

Comprehensive Evaluation of the Efficiency of Yeast Cell Wall Extract to Adsorb Ochratoxin A and Mitigate Accumulation of the Toxin in Broiler Chickens.

Ochratoxin A (OTA) is a common mycotoxin contaminant in animal feed. When absorbed from the gastrointestinal tract, OTA has a propensity for pathological effects on animal health and deposition in animal tissues. In this study, the potential of yeast cell wall extracts (YCWE) to adsorb OTA was evaluated using an in vitro method in which consecutive animal digestion events were simulated. Low pH markedly increased OTA binding to YCWE, which was reversed with a pH increased to 6.5. Overall, in vitro analysis revealed that 30% of OTA was adsorbed to YCWE. Additional computational molecular modelling revealed that change in pH alters the OTA charge and modulates the interaction with the YCWE β-d-glucans. The effectiveness of YCWE was tested in a 14-day broiler chicken trial. Birds were subjected to five dietary treatments; with and without OTA, and OTA combined with YCWE at three dosages. At the end of the trial, liver OTA deposition was evaluated. Data showed a decrease of up to 30% in OTA deposits in the liver of broilers fed both OTA and YCWE. In the case of OTA, a tight correlation between the mitigation efficacy of YCWE between in vitro and in vivo model could be observed.

Amelioration of ochratoxin-A induced cytotoxicity by prophylactic treatment of N-Acetyl-L-Tryptophan in human embryonic kidney cells

Ochratoxin A (OTA) is known to induce nephro-toxicity via induction of cellular redox homeostasis perturbation, mitochondrial hyperpolarisation and depolarization, protein synthesis inhibition leading to apoptosis. In the present study, protective efficacy of N-Acetyl-L-Tryptophan (NAT) against OTA induced toxicity was evaluated using Human Embryonic Kidney (HEK-293) cells. Cells were treated with NAT (0–200 μg/ml) before OTA treatment (0–20 μg/ml) and protective efficacy of NAT was evaluated using MTT and SRB assay. OTA-induced intracellular ROS generation and its inhibition by NAT (2.5 μg/ml) pre-treatment was evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe. Effects of NAT pre-treatment on OTA treated cells were also evaluated in terms of cell cycle perturbations and mitochondrial membrane potential disturbance using flowcytometry. Results of the study demonstrated significant (∼89 % cell growth in comparison to 50% in OTA alone group; P < 0.05) protection by NAT to the HEK-293 cells against OTA mediated cell death in terms of cell viability. Further, significant reduction in ROS levels and mitochondrial membrane potential disturbance was also observed in NAT pre-treated plus OTA cells as compared to only OTA treated cells. Significant (p < 0.05) arrest in G0 and S phase of cell cycle was observed in OTA treated cells that was found to be inhibited by NAT pre-treatment to OTA treated cells. Also, molecular docking analysis demonstrated higher probability of NAT to bind with OTA binding pocket on phenylalanyl t-RNA synthetase, resulting in inhibition of OTA incorporation in the newly synthesized peptides and thus may ameliorate OTA induced protein synthesis inhibition. Conclusively, present study suggested that NAT offers protection against OTA toxicity in HEK-293 cells by counterbalancing oxidative stress, cell cycle regulation, mitochondrial membrane potential stabilization, protein synthesis inhibition and cell death retardation.

Detachable nanoladders: A new method for signal identification and their application in the detection of ochratoxin A (OTA)

A highly sensitive fluorescence turn-off biosensor for the detection of ochratoxin A (OTA) was developed based on graphene oxide (GO) and an aptamer-induced detachable nanoladders. In this assay, two types of ssDNA (H1 and H2) were involved in the assembly of the DNA nanoladders, in which H1 was labeled with fluorophore, and H2 was the OTA binding aptamer. Self-assembly of the DNA nanoladders with the addition of GO weakened its adsorption and the fluorescence intensity remained strong. In the presence of OTA, the aptamer was specifically recognized and an aptamer-OTA complex was formed, leading to the detached of DNA nanoladders. With the addition of GO, the released H1 was adsorbed on the GO surface, thus efficiently quenching the fluorescence signal (turning off). The detection limit of OTA in this assay was 4.59 nM. To improve the sensitivity of this strategy, we creatively developed an alternative strategy to replace the sturdy nanoladders with frail nanoladders. More precisely, the sequences of H1 had mismatched bases, which, when hybridized with H2 could be used to create long non-perfect complementary nanoladders. For the mismatched bases-based frail nanoladders, it was easier for OTA to bind its aptamer sequence, thus enabling a more thorough and faster detachment of the nanoladders, along with a greater degree of fluorescence quenching. The detection limit for OTA was estimated to be 0.1 nM. The biosensors we developed were sensitive, specific, enzyme-free, cost-effective and can be applied in red wine samples spiked with known concentration of OTA.