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Characterization of biotinylated M13OTA phage. (A) The confocal laser microscope images of streptavidin-polystyrene microspheres after incubating with streptavidin-FITC and biotinylated M13OTA phage; (B) The confocal laser microscope images of streptavidin-polystyrene microspheres after incubating with streptavidin-FITC and unmodified M13OTA phage; (C) Optimization of biotin
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Conventional enzyme-linked immunosorbent assay (ELISA) is commonly used for Ochratoxin A (OTA) screening, but it is limited by low sensitivity and harmful competing antigens of enzyme-OTA conjugates. Herein, a bifunctional M13 bacteriophage with OTA mimotopes fused on the p3 protein and biotin modified on major p8 proteins was introduced as an eco-...
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... the fluorescence property of the used CdTe QDs was evaluated to select the optimal parameters of the signal transduction system. CdTe QDs exhibit the maximum emission peak at about 590 nm ( Figure S2). The effect of pH on the fluorescence of the CdTe QDs was examined, given that fluorescence intensity is susceptible to the concentration of hydrogen ions in a solution ( Figure S3). ...
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... verify the avidin-specific binding ability of the resultant biotinylated M13OTA phage, it was used as a bridge for conjugating streptavidin-modified polystyrene (PS) micro-spheres with streptavidin-FITC. Laser confocal microscopy reveals a bright green fluorescence on the surface of the microsphere (Figure 2A). Conversely, unmodified M13OTA phage cannot form a bridge between streptavidin-FITC and the microsphere ( Figure 2B). ...
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... confocal microscopy reveals a bright green fluorescence on the surface of the microsphere (Figure 2A). Conversely, unmodified M13OTA phage cannot form a bridge between streptavidin-FITC and the microsphere ( Figure 2B). Thus, the as-prepared biotinylated M13OTA phage can achieve high-density enzyme loading through the biotin-avidin system. ...
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... were conducted to develop "antibodyphage-GOx" sandwich structures on anti-OTA ascitic fluid-modified microplates, and the fluorescence intensity of the subsequently added CdTe QDs were detected to evaluate the performance of biotinylated M13OTA phage. The results in Figure 2C demonstrate that the fluorescence quenching rate increases as the biotin-to-phage dosage ratio in the phage increases and reaches a plateau at a dosage ratio of 80,000:1. Therefore, biotinylated M13OTA phage developed at the ratio of 80,000:1 saturates the loading capacity of the phage. ...
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... results illustrated in Figure S6 reveal that each biotinylated phage can bind to approximately 269 of streptavidin molecules, indicating the high loading capacity of biotin-GOx for signal amplification. Then, the performance of the bifunctional phage acting as a competing antigen in the proposed FLISA was assessed by parallelly conducting three assays ( Figure 2D). In the blank control assay, no GOx was captured on the microplates because of the absence of the bifunctional phage, thus leading to an unchanged fluorescence intensity of CdTe QDs. ...
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Ochratoxin A (OTA) is considered the most toxic member of the ochratoxin group. Herein, a novel label-free electrochemical sensor based on the horseradish peroxidase (HRP) enzyme is developed for OTA detection. The HRP enzyme was covalently immobilized on the working electrode of a planar boron-doped diamond (BDD) electrochemical microcell previous...
Citations
... To further enhance the detection sensitivity, these immunosensor mimotopes can be labeled with biotin or composite probes. In a noteworthy study by Tong, Weipeng, et al. (107), a simulated peptide was obtained from a commercial phage display library, and to bolster sensitivity, biotin was introduced as a competitive antigen by modifying the main protein. The proposed method exhibited excellent linear detection within the range of 4.8 to 625 pg/mL, boasting an impressive detection limit (LOD) of 5.39 pg/ mL. ...
Cholera, a persistent global public health concern, continues to cause outbreaks in approximately 30 countries and territories this year. The imperative to safeguard water sources and food from Vibrio cholerae, the causative pathogen, remains urgent. The bacterium is mainly disseminated via ingestion of contaminated water or food. Despite the plate method’s gold standard status for detection, its time-consuming nature, taking several days to provide results, remains a challenge. The emergence of novel virulence serotypes raises public health concerns, potentially compromising existing detection methods. Hence, exploiting Vibrio cholerae toxin testing holds promise due to its inherent stability. Immunobiosensors, leveraging antibody specificity and sensitivity, present formidable tools for detecting diverse small molecules, encompassing drugs, hormones, toxins, and environmental pollutants. This review explores cholera toxin detection, highlighting phage display-based nano immunosensors’ potential. Engineered bacteriophages exhibit exceptional cholera toxin affinity, through specific antibody fragments or mimotopes, enabling precise quantification. This innovative approach promises to reshape cholera toxin detection, offering an alternative to animal-derived methods. Harnessing engineered bacteriophages aligns with ethical detection and emphasizes sensitivity and accuracy, a pivotal stride in the evolution of detection strategies. This review primarily introduces recent advancements in phage display-based nano immunosensors for cholera toxin, encompassing technical aspects, current challenges, and future prospects.
... To the best of our knowledge, this is the most sensitive ELISA that has been achieved so far based on this newly produced mAb. In should be addressed that some aptamer (or Phage)-based ELISAs or sensors may obtain high sensitivity for OTA detection [37][38][39][40]. In the last decades, many ELISAs for the detection of OTA in food samples based on pAb or mAb have been developed [23][24][25][26][27][28][29][30][31][32][33][34][35][36]. ...
... To the best of our knowledge, this is the most sensitive ELISA that has been achieved so far based on this newly produced mAb. In should be addressed that some aptamer (or Phage)-based ELISAs or sensors may obtain high sensitivity for OTA detection [37][38][39][40]. ...
In this study, an extremely highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a newly produced monoclonal antibody (mAb) for the detection of ochratoxin A (OTA) in food samples was developed. OTA-Bovine serum albumin (BSA) conjugate was prepared and used as the immunogen for the production of the mAb. Among four hybridoma clones (8B10, 5C2, 9B7, and 5E11), the antibody from 8B10 displayed the highest affinity recognition for OTA. Based on the mAb (8B10), the IC50 and LOD of the ELISA for OTA were 34.8 pg mL−1 and 1.5 pg mL−1, respectively, which was 1.53~147 times lower than those in published ELISAs, indicating the ultra-sensitivity of our assay. There was no cross-reactivity of the mAb with the other four mycotoxins (AFB1, ZEN, DON, and T-2). Due to the high similarity in molecular structures among OTA, ochratoxin B (OTB), and ochratoxin C (OTC), the CR values of the mAb with OTB and OTC were 96.67% and 22.02%, respectively. Taking this advantage, the ELISA may be able to evaluate total ochratoxin levels in food samples. The recoveries of the ELISA for OTA in spiked samples (corn, wheat, and feed) were 96.5–110.8%, 89.5–94.4%, and 91.8–113.3%; and the RSDs were 5.2–13.6%, 8.2–13.0%, and 7.7–13.7% (n = 3), respectively. The spiked food samples (corn) were measured by ELISA and HPLC-FLD simultaneously. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation of y = 0.918x − 0.034 (R2 = 0.985, n = 5) was obtained. These results demonstrated that the newly produced mAb-based ELISA was a feasible and ultra-sensitive analytical method for the detection of OTA in food samples.
... A comparison of our work with previously reported immunoassays based on mimotope (phagedisplayed mimotope peptide or Anti-Idiotypic nanobody) and conventional conjugate with Nb28 for OTA was listed in Table S4. The detection speed of APN-ELISA is superior to most methods, while its sensitivity is comparable with most reported conventional immunoassays, such as ELISA, chemiluminescent ELISA, fluorescent ELISA, and lateral flow immunoassay (Song, Feng, Leng, Huang, Fang, Tong, et al., 2021;Tong, Fang, Xiong, Wei, Leng, Hu, et al., 2021;Xu, He, He, Qiu, Chen, Chen, et al., 2014;You, Luo, Hu, Xu, Guo, & He, 2020). The PN-ELISA is more sensitive than the majority of reported immunoassays while showing comparable detection speed with most methods, such as ELISA, fluorescent ELISA, and electrochemical immunosensor (Hou, Ma, Meng, Xu, & He, 2019;Song, Feng, Leng, Huang, Fang, Tong, et al., 2021;Tong, Fang, Xiong, Wei, Leng, Hu, et al., 2021). ...
... The detection speed of APN-ELISA is superior to most methods, while its sensitivity is comparable with most reported conventional immunoassays, such as ELISA, chemiluminescent ELISA, fluorescent ELISA, and lateral flow immunoassay (Song, Feng, Leng, Huang, Fang, Tong, et al., 2021;Tong, Fang, Xiong, Wei, Leng, Hu, et al., 2021;Xu, He, He, Qiu, Chen, Chen, et al., 2014;You, Luo, Hu, Xu, Guo, & He, 2020). The PN-ELISA is more sensitive than the majority of reported immunoassays while showing comparable detection speed with most methods, such as ELISA, fluorescent ELISA, and electrochemical immunosensor (Hou, Ma, Meng, Xu, & He, 2019;Song, Feng, Leng, Huang, Fang, Tong, et al., 2021;Tong, Fang, Xiong, Wei, Leng, Hu, et al., 2021). Compared with the indirect competitive ELISA based on Nb28 and conventional conjugate, the PN-ELISA shows higher sensitivity, while the APN-ELISA shows comparable sensitivity and detection speed. ...
Mimotope-based immunoassays for mycotoxins eliminate the requirement for large amounts of mycotoxin standards for the chemosynthesis of artificial antigens. Herein, the nanobody-based magnetic beads were used to screen the mimotope (peptidomimetic) of ochratoxin A (OTA) from the phage-displayed peptide library. The interactions between nanobody and the most sensitive Y4 peptidomimetic were investigated by computer-assisted simulation and compared with those between nanobody and OTA. By combining the nanobody, the phage-displayed Y4 and alkaline phosphatase-tagged Y4 fusion protein as the competing antigens, were used to develop two novel immunoassay platforms (PN-ELISA and APN-ELISA). The two methods are advantageous in the use of nontoxic substitutes of OTA and avoiding the use of monoclonal antibodies. Moreover, good analytical performances of both methods were obtained and confirmed by liquid chromatography tandem mass spectrometry. Therefore, the proposed novel methods based on nanobody and peptidomimetic were demonstrated to be highly reliable for detecting OTA in food.
... Many methods have been used to detect OTA, including traditional methods such as high-performance liquid chromatography (HPLC), high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS), thin-layer chromatography (TLC), and enzyme-linked immunosorbent assay (ELISA) [11][12][13][14]. These methods have shown good accuracy and sensitivity, but their disadvantages such as expensive equipment, time-consuming procedures, the need for specialized personnel, and antibody fallibility have limited their application. ...
A novel three-dimensional (3D) porous nitrogen-sulfur co-doped carbon (N–S–C) mesh was synthesized and used for the first time as the quenching material to construct a fluorescent aptasensor for ochratoxin A (OTA) detection. The fluorescent aptasensor with enzyme-free signal amplification strategy was developed by using cDNA as a promoter to trigger hybridization chain reaction (HCR), which effectively improved the sensitivity of this aptasensor. In the absence of OTA, 3D porous N–S–C mesh can adsorb carboxyfluorescein FAM-labeled hairpin DNA1 (H1-FAM) and hairpin DNA2 (H2) and quench the fluorescence of FAM. In the presence of the OTA, the OTA specifically binds to the aptamer strand and the DNA duplex undergoes dissociation. The released cDNA in turn serves as a promoter for HCR, and the strand assembly of H1-FAM and H2 is triggered by the promoter to generate long-strand DNA polymers via HCR, resulting in an increasing fluorescent signal. Under optimal conditions, there was a good linear relationship between lgCOTA and fluorescence intensity difference in the range 0.01–500 ng/mL (R² = 0.993), and the detection limit was 2.7 pg/mL. The designed sensor platform was applied to determine spiked OTA in peanut, wheat flour, corn flour, black tea, and wine with recoveries in the range of 94.4–119.6%.
Graphical Abstract
... The abundant copies of p8 proteins can be extensively functionalized as a container for high-density loading of signal transducers or regulators (e.g., fluorescent dyes and enzymes), resulting in remarkably enhanced signal intensities of biosensors [34]. Previously, we adopted an OTA-mimicking M13 phage (M13OTA) integrated with an OTA mimotope at the p3 proteins as an enzyme container to improve the sensitivity of a fluorescent ELISA for OTA detection [35]. ...
... Moreover, numerous biotin molecules were modified on the major p8 proteins. According to our previous report, the as-prepared bio-M13OTA exhibits a high loading capacity of 269 streptavidin molecules [35]. This finding indicates that biotin-M13OTA can be used as the enzyme container for carrying a mass of biotinylated GOx (biotin-GOx). ...
“Point of care” (POC) methods without expensive instruments and special technicians are greatly needed for high-throughput analysis of mycotoxins. In comparison, the most widely used screening method of the conventional enzyme-linked immunosorbent assay (ELISA) confronts low sensitivity and harmful competing antigens. Herein, we develop a plasmonic-photothermal ELISA that allows precise readout by color-temperature dual-modal signals based on enzymatic reaction-induced AuNP aggregation for highly sensitive detection of ochratoxin A (OTA). The bifunctional M13 phage carrying OTA that mimics the mimotope on the end of p3 proteins and abundant biotin molecules on the major p8 proteins is adopted as an eco-friendly competing antigen and enzyme container for amplifying the signal intensity. Under optimal conditions, both colorimetric and photothermal signals enable good dynamic linearity for quantitative OTA detection with the limits of detection at 12.1 and 8.6 pg mL−1, respectively. Additionally, the proposed ELISA was adapted to visual determination with a cutoff limit of 78 pg mL−1 according to a vivid color change from deep blue to red. The recoveries of OTA-spiked corn samples indicate the high accuracy and robustness of the proposed method. In conclusion, our proposed strategy provides a promising method for eco-friendly and sensitive POC screening of OTA. Moreover, it can be easily applied to other analytes by changing the involved specific mimotope sequence.
... Besides, the sensitivity of immunoassays is greatly affected by the signal probes. To date, many fluorescent probes (e.g., quantum dots (QDs), up-conversion nanoparticles, and lanthanide ions) instead of traditional enzymes have been applied to enhance the sensitivity of immunoassays [28][29][30][31][32][33][34][35]. Among them, QDs are ideal fluorescent materials because of their narrow emission spectra, broad excitation, high fluorescent intensity, and high stability [36,37]. ...
In this study, a quantum-dot-bead (QB)-based fluorescence-linked immunosorbent assay (FLISA) using nanobodies was established for sensitive determination of the Cry2A toxin in cereal. QBs were used as the fluorescent probe and conjugated with a Cry2A polyclonal antibody. An anti-Cry2A nanobody P2 was expressed and used as the capture antibody. The results revealed that the low detection limit of the developed QB-FLISA was 0.41 ng/mL, which had a 19-times higher sensitivity than the traditional colorimetric ELISA. The proposed assay exhibited a high specificity for the Cry2A toxin, and it had no evident cross-reactions with other Cry toxins. The recoveries of Cry2A from the spiked cereal sample ranged from 86.6–117.3%, with a coefficient of variation lower than 9%. Moreover, sample analysis results of the QB-FLISA and commercial ELISA kit correlated well with each other. These results indicated that the developed QB-FLISA provides a potential approach for the sensitive determination of the Cry2A toxin in cereals.
An accurate and sensitive competitive enzyme-linked immunosorbent assay (ELISA) based on persistent luminescence nanoparticles Zn2GeO4:Mn2+, Eu3+ (ZGME) was developed for detecting ochratoxin A (OTA), a powerfully toxic mycotoxin usually found in grains. As a signal output element of autofluorescence-free biosensors, ZGME can be integrated into ELISA with glucose oxidase (GOx)-binding OTA molecules due to its excellent pH-responsive persistent luminescence. In the absence of OTA, the OTA-GOx conjugate was captured by the anti-OTA monoclonal antibody (anti-OTA mAb) pre-coated on the 96-well plate. The results indicate a decrease in the pH value of the solution, which triggered the quenching of ZGME luminescence due to GOx-dependent gluconic acid production. The presence of OTA inhibited the binding of OTA-GOx on the plate, thus decreasing the production of gluconic acid and increasing the persistent luminous intensity of ZGME. Under the optimized concentrations of anti-OTA mAb and OTA-GOx, quantitative determination of OTA was achieved by plotting the increase or decrease in persistent luminescence intensity of ZGME at 535 nm. In this study, the linear range was from 0.1 μg L-1 to 63 μg L-1, and the limit of detection (LOD) was as low as 0.045 μg L-1. In five food samples (corn grit, brown rice, soybean, rice, and wheat), the results exhibited good stability and repeatability, with a recovery range from 81.3% to 94.4% and a relative standard deviation (RSD) of less than 4.2%. Hence, the established method provides a sensitive, accurate, and autofluorescence-free approach for the determination of OTA in different grain samples.
