hazardous materials magazine
August 5, 2023
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Bongkrekic acid (BA) is a mitochondrial toxin that causes high mortality but is often misclassified as other food poisoning. The BA immunoassay remains challenging due to the unavailability of specific antibodies. In this work, a monoclonal antibody against BA was first generated and a two-module immunosensor was established for field and laboratory detection. The antibody showed good affinity (Kd=0.33 μM) and sensitivity (IC50=17.9 ng/mL (in ELISA), negligible cross-reactivity with common mycotoxins. Under double module conditions, the fluorometric test (FA) was performed based on the internal filtering effect of carbon dots (CD) and oxidized 3,3′,5,5′-tetramethylbenzidine (TMB), while the colorimetric test (CA ) was performed using TMB -And2+-mediated rapid surface etching of gold nanostars (Au NS). The proposed immunosensor showed good sensitivity and reproducibility for BA in food samples with a detection limit below 10 ng/mL and recoveries ranging from 80.0% to 103.6%, in good agreement with standard LC-MS/MS consistency. Overall, the proposed immunosensor is an ideal tool for screening BA contaminants in food with good sensitivity and high efficiency.
Bongkrekinic acid (BA) is a mitochondrial toxin that was first discovered in 1930 by the Dutch scientists Mertens and Vanveen , . Since 1975, consumption of infected tempe bongkrek has resulted in nearly 3000 cases of BA poisoning, including at least 150 deaths . Recently, a family in Jixi City, Heilongjiang Province, China died from eating corn flour contaminated with BA. However, it was initially misidentified as a food poisoning event caused by aflatoxins , . Since then, the harmfulness of BA has been gradually emphasized. BA is secreted by the pathogenic variantBurkholderia Gladiolus PV.cocovenenans, which can easily contaminate fermented grain products, scabies and fungi. It mainly acts on the respiratory chain system and blocks the exchange of adenosine diphosphate and adenosine triphosphate between the inner membranes of mitochondria causing poisoning . The incubation period of BA in the human body is 1-10 hours, and the oral median lethal dose is 3.16 mg/kg. Moreover, the mortality rate of BA exceeds 40%, which is much higher than other common bacterial food poisonings , . Therefore, monitoring BA is essential for food safety and human health.
Previously, instrumental methods such as high-performance liquid chromatography-mass spectrometry were the main analytical methods for BA detection , . However, these methods have the disadvantage of requiring expensive reagents, specialized equipment, and skilled personnel. Recently, Zhang et al.Zhang i sur., ($godina$) reported a UV-Vis and APP dual-mode detection platform for BA with a detection limit of only 6.0 ng/mL based on the unique optical properties of gold nanoparticles and the electrostatic interaction between gold nanoparticles and BA. However, this method has the limitations of false positive results and low specificity. In this sense, immunoassays that specifically recognize antigens and antibodies can be a potential method for monitoring BA due to their high sensitivity, specificity and accessibility , . Colorimetry (CA) has inherent advantages in convenience and visual detection, but its low sensitivity limits its application in monomodal reading. In fact, noble metal nanostructures have been extensively studied in recent years for the development of naked-eye colorimetric sensors without the use of complex devices , . Fluorescence assays (FA), which mainly rely on intrinsic velocity effects, show higher sensitivity and accuracy than conventional colorimetric sensors , . As for FA, fluorescent probes are closely related to the sensitivity of the method. At the same time, carbon dots (CDs) have also attracted much attention in the past decade due to their high photoluminescence quantum yield, abundance of raw materials on earth, low production cost, and environmentally friendly preparation. They are ideal fluorescent materials. FA probe (). Recently, the dual immunosensor has quietly entered the market to meet the precision and sensitivity requirements of quantitative analysis, has the advantages of colorimetric or fluorescence methods, and can use dual-channel signals as self-control to prevent oscillations , , .
To our knowledge, the anti-BA antibody obtained in this work is the first. Typically, monoclonal antibodies (mAbs) against BA with good sensitivity and specificity are prepared by immunogen optimization and hybridoma selection. Based on this, a fluorescent and colorimetric two-module immunosensor based on red CDs and gold nanostars (Au NS) was established for on-site and laboratory detection of BA. In Scheme 1, 3,3',5,5'-tetramethylbenzidine (TMB) was oxidized by horseradish peroxidase to oxTMB, which overlapped well with the fluorescence emission spectrum (650 nm) of CD. The regularity of the fluorescence quenching is caused by the initiation of internal velocity effects. Simultaneously, the oxidation products released by oxTMB etched the Au NSs to form colored products for visual inspection. In this study, the developed immunosensor demonstrated excellent detection performance for BA by simultaneously combining fluorescent and visual signals, paving the way for sensitive and rapid detection in the field.
BA standards were provided by ANPEL Laboratory Technologies Inc (Shanghai, China). Bovine serum albumin, lactoferrin (LF), and ovalbumin (OVA) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). TMB, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)->3-ethylcarbodiimide hydrochloride and sodium citrate were purchased from Aladdin of Co. Ltd (China Shanghai). Hydrogen peroxide (H2Europa2), sulfuric acid (H2so4), hydrochloric acid, sodium hydroxide and urea
Characterization of artificial antigens
Collect UV-vis spectra of the final bound protein and the carrier protein to confirm successful conjugation of the hapten to the carrier protein. Figure S1 shows the characteristic absorption peak of BA at 287 nm, while OVA and LF show maximum absorption peak at around 220 nm. BA-OVA and BA-LF showed a specific absorbance maximum at 230 nm, reflecting the identity of the carrier protein and a change in absorbance with clear absorption compared to BA. Therefore, the UV-Vis spectrum is
In conclusion, a cell line capable of secreting high-quality mAb (B9) against BA was first selected. Then, a representative two-module mAb-based immunoassay was constructed for BA detection with CD fluorescence intensity and blue shift LSPR of Au NS. An ingenious combination of TMB2+Etching Au NSs and CDs extinction gives the test better capabilities, including multimodal signal output and high sensitivity. The established two-module immunoassay showed a LOD of
impact on the environment
Bongkrekinic acid (BA) is a mitochondrial toxin secreted by a pathogenic variantBurkholderia Gladiolus PV. Kokoidan(BGC), wine-fermented grain products that are easily contaminated, white mold, fungus, etc., and the mortality rate is as high as 50%. However, the most commonly used BA detection methods are instrumental methods, and fast and sensitive BA detection methods are still a challenge. In this work, we established a monoclonal antibody (mAb) with good selectivity for BA for
CrediT Author Contribution Statement
Xue-Ming Cao:Conceptualization, methodology, data analysis, writing - manuscript.Li-Hua Li:Conceptualization, formal analysis, research.Hong-zhi Liang:Methodology, Validation.Jia-Dong Li:Program management, data analysis.Zi-Jian Chen:Program management, data analysis.Lin Luo:validation, methodology.Yi-Na Lu:method.Yu-Xin Zhong:software, resources.Yu-Dong Shen:Supervision and investigation.Hong Taolei:Fundraising, monitoring.Wang Hong:Writing - overview i
Statement of competing interests
The authors declare that they have no known financial interests or personal relationships that could influence the work reported in this article.
this paper receivedNational Natural Science Foundation of China(32061160473), ovoLighthouse Labs technology project, Depth (DT20220033), iShantou Customs Technology Project, Depth (2022STK001).
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