由蒽醌衍生的二价和三价过渡金属偶氮配合物及其致突变－畸变作用

制备了4-(5′-氨基萘-3′-磺酸)偶氮-(2″-N-(4苁,6苁-二氯-S-三嗪)苯-5″-(β-羟乙基砜硫酸酯)-2-甲基蒽醌(2a),4-(2′-氨基苯磺酸)偶氮-(2″-N-(4苁,6苁-二氯-S-三嗪)-3″-苯磺酸)-2-甲基蒽醌,4-(5′-氨基萘-3′-磺酸)偶氮-(2″-N-(4苁,6苁-二氯-S-三嗪)苯-5″-(β-羟乙基砜硫酸酯)-2-甲基蒽醌和4-(p-(β-羟乙基砜硫酸酯)偶氮-(2″-N-(4苁,6苁-二氯-S-三嗪)萘-5″-磺酸)-2-甲基蒽醌的FeIII,CoII金属配合物,并在安姆/沙门氏菌/微粒体致突变试验(Ames/Salmonella/Microsome Test)和大鼠胚胎中测定其致突变-畸变作用。不管是否使用代谢赋活剂S9mix,2a-Fe和2a-Co对TA98 and TA100菌株均无致突变-畸变作用。     

硒化螺旋藻对实验诱发大鼠肝癌过程的作用

目的:观察硒化螺旋藻(Se-spirulina)对黄曲霉毒素B1(AFB1)致肝癌作用的影响。方法:实验动物按随机区域分组法分为A、B、C组,每组15只,A、B、C组分别为高剂量组、低剂量组、对照组,各组动物腹腔注射AFB1两周,A、B组大鼠在接受AFB1期间分别喂食含量分别为9.3×10-3和2.3×10-3的硒化螺旋藻混合饲料,8周后处死动物,观察各组动物肝组织内γ-谷氨酰转肽酶阳性肝细胞增生灶(-γGT灶)的数量(个/cm2)和大小(mm2/个)。结果:高、低剂量硒化螺旋藻均能抑制AFB1诱发的-γGT灶的数量和大小,高剂量组、低剂量组-γGT灶的数量分别为(0.90±1.07)个/cm2和(3.72±4.72)个/cm2,均低于对照组(6.10±6.30)个/cm2,高剂量组与对照组相比有显著意义(P<0.05),抑制率分别为85%和39%;高剂量组、低剂量组-γGT灶大小分别为(0.24±0.28)mm2/个和(1.94±2.32)mm2/个,均低于对照组(2.36±3.76)mm2/个,但无显著意义(P>0.05),抑制率分别为90%和17%。结论:硒化螺旋藻有抑制AFB1诱发大鼠肝癌前病变的作用,而高剂量硒化螺旋藻的抑制作用更优。     

Voltammetric Determination of Aflatoxin B1

For the first time the possibility of voltammetry used for the determination of aflatoxin B1 on a glassy carbon electrode was shown. The effect of pH of a supporting electrolyte on the analytical signal of aflatoxin B1 has been investigated and it was shown that there is a more pronounced peak with a maximum current at pH of 5.33. The most favorable supporting electrolyte for a linear range of detectable concentrations of aflatoxin B1 – 0.1 M (NH₄)₂SO₄ was determined. The results of research on the development of conditions of voltammetric measurement of aflatoxin B1 are presented.     

自制混合型固相萃取柱-高效液相色谱法同时测定食品中黄曲霉毒素B1、M1

建立了基于自制混合型固相萃取柱的样品净化/高效液相色谱测定食品中黄曲霉毒素B1、M1的方法。样品经60%乙腈水溶液提取、离心后,通过自制固相萃取柱排除杂质干扰,流出液以Shim-pack VP-ODS C18色谱柱为分离柱,水和乙腈为流动相,用荧光检测器检测,外标法定量。考察了柱类型、柱容量、取样量、提取溶液和流速等对检测的影响,优化了实验条件。在优化条件下,2种毒素在0.40～100μg/L质量浓度范围内与峰面积呈良好的线性关系,相关系数为0.999 4～0.999 7,检出限(S/N=3)为0.050μg/kg。在样品中分别加入0.40、1.0、100μg/L 3种浓度水平的标准品,其加标回收率为53%～112%,相对标准偏差为2.7%～7.1%。该法灵敏度高,操作简单﹑快速,适用于花生、开心果和奶粉等食品中痕量黄曲霉毒素B1和M1的测定。     

Trichoderma Enzymes for Degradation of Aflatoxin B1 and Ochratoxin A

The contamination of agricultural products with mycotoxins causes risks to animal and human health and severe economic losses. Mycotoxicoses can be reduced by preventing fungal infection using chemical and biological approaches. The chemical strategies can release toxic molecules; therefore, strategies for biological control are being evaluated, such as using nontoxic fungi and their metabolites. This work evaluated the effect of exoenzymes produced by the beneficial fungus Trichoderma afroharzianum strain T22 in degrading Aflatoxin B1 (AFB1) and Ochratoxin A (OTA). The ability of Trichoderma to produce hydrolases was stimulated by using different inducing substrates. The highest AFB1 and OTA degradation activity was obtained using a medium containing lyophilized mushrooms and crude fiber. The T. afroharzianum T22’s ability to reduce mycotoxins may be attributed to peroxidase enzymes. This study showed that T. afroharzianum strain T22 or its peroxidase supplementation could represent a sustainable strategy for the degradation of AFB1 and OTA in feed and food products.     

A Guide to Thin-Layer Chromatographic Systems for the Separation of Aflatoxin B1, B2, G1 and G2

Abstract

The separation of aflatoxin B₁, B₂, G₁ and G₂ was compared on six commercial silica gel plates in twelve solvent systems. Two of the solvent systems, chloroform: acetone: ammonium hydroxide (90: 10: 0.25) and chloroform: acetone: hexane (85: 15: 20) resolved the four aflatoxins on all the tested plates. The solvent modifier played an important role in the resolution of these compounds. The effect of the hardness of the plate is also discussed.     

一种简单灵敏的基于适配体的黄曲霉毒素B1电化学传感器

黄曲霉毒素B1（AFB1）以其高毒性和致癌性成为食品安全隐患而备受关注. 本文拟构建一种新颖、简单、快速、灵敏的传感器用于谷物食品中AFB1的痕量检测. 将介孔碳（CMK）修饰在工作电极表面来增大电极的表面积,再将工作电极恒电位沉积金纳米粒子（AuNPs）,提高电信号的同时,为下一步巯基化适配体的连接提供位点. 检测过程中,AFB1可以竞争性地去除吸附在适配体链上的亚甲基蓝（MB）引起电信号的变化,对AFB1进行定量检测. 修饰的工作电极导电性能得到改善,灵敏度大大提高,对AFB1的线性响应范围为0.1 ~ 75 μg·L^-1,检出限低至36 ng·L^-1. 在对不同谷物食品（大米、玉米、糯米）进行加标回收实验中,回收率在92.3% ~ 103.6%范围之间,实现对目标物的定量检测. 本文为食品中AFB1快速检测方法提供了一种新思路和新方法.     

适配体生物传感器在黄曲霉毒素B1检测中的应用

该文首先对黄曲霉毒素B1（AFB1）的相关性质及其传统检测方法进行了介绍,随后概述了近年来基于光学、电化学以及微流控芯片的适配体生物传感器的构建及其在AFB1检测领域中的应用,旨为适配体生物传感器的实际应用提供参考;并通过探讨目前开发的检测方法存在的问题,对适配体生物传感器前景和未来趋势进行了展望。     

A Simple Structure-Switch Aptasensor Using Label-Free Aptamer for Fluorescence Detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is one of the mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus, and it causes contamination in foods and great risk to human health. Simple sensitive detection of AFB1 is important and demanded for food safety and quality control. Aptamers can specifically bind to targets with high affinity, showing advantages in affinity assays and biosensors. We reported an aptamer structure-switch for fluorescent detection of aflatoxin B1 (AFB1), using a label-free aptamer, a fluorescein (FAM)-labeled complementary strand (FDNA), and a quencher (BHQ1)-labeled complementary strand (QDNA). When AFB1 is absent, these three strands assemble into a duplex DNA structure through DNA hybridization, making FAM close to BHQ1, and fluorescence quenching occurs. In the presence of AFB1, the aptamer binds with AFB1, instead of hybridizing with QDNA. Thus, FAM is apart from BHQ1, and fluorescence increases with the addition of AFB1. This assay allowed detection of AFB1 with a detection limit of 61 pM AFB1 and a dynamic concentration range of 61 pM to 4 μM. This aptamer-based method enabled detection of AFB1 in complex sample matrix (e.g., beer and corn flour samples).     

柱前衍生-高效液相色谱法测定茶叶中的黄曲霉毒素B1

应用柱前衍生-高效液相色谱法测定茶叶中黄曲霉毒素B1的含量。样品采用乙腈(85+15)溶液提取,滤液用MycoSepTM226柱净化,加入正己烷和三氟乙酸衍生,经C18色谱柱分离,荧光检测器检测。黄曲霉毒素B1的质量浓度在0.20~10.0μg·L-1范围内与其峰面积呈线性关系,检出限(3S/N)为0.1μg·kg-1。在0.5,1.0,5.0μg·L-1等3个浓度水平进行加标回收试验,回收率在91.9%~102%之间,测定值的相对标准偏差(n=6)在1.5%~6.9%之间。     

黄曲霉素B1在银团簇表面吸附的表面增强拉曼光谱

采用密度泛函理论(DFT)的B3LYP方法和6-311g(d, p)(C, H, O)/LanL2DZ(Ag)基组, 优化得到黄曲霉素分子AFB₁与Ag小团簇形成的复合物AFB₁-Ag_n (n=2, 4, 6)的稳定结构, 并计算了三种复合物的表面增强拉曼光谱(SERS)和预共振拉曼光谱(SERRS), 与实验结果相一致. 计算结果显示: 三种复合物表面增强拉曼光谱中C＝O伸缩振动模的增强因子约为10²-10³, 是由于极化率改变引起的静化学增强. 根据含时密度泛函理论(TDDFT)方法计算得到的吸收光谱, 分别选择407.5、446.2和411.2 nm作为入射光, 计算三种复合物的共振拉曼光谱, 发现在SERRS光谱中, Ag―O伸缩振动的增强因子达到10⁴量级, 主要是由电荷转移产生的共振增强引起的.     

Virtual Extensive Read-Across: A New Open-Access Software for Chemical Read-Across and Its Application to the Carcinogenicity Assessment of Botanicals

Read-across applies the principle of similarity to identify the most similar substances to represent a given target substance in data-poor situations. However, differences between the target and the source substances exist. The present study aims to screen and assess the effect of the key components in a molecule which may escape the evaluation for read-across based only on the most similar substance(s) using a new open-access software: Virtual Extensive Read-Across (VERA). VERA provides a means to assess similarity between chemicals using structural alerts specific to the property, pre-defined molecular groups and structural similarity. The software finds the most similar compounds with a certain feature, e.g., structural alerts and molecular groups, and provides clusters of similar substances while comparing these similar substances within different clusters. Carcinogenicity is a complex endpoint with several mechanisms, requiring resource intensive experimental bioassays and a large number of animals; as such, the use of read-across as part of new approach methodologies would support carcinogenicity assessment. To test the VERA software, carcinogenicity was selected as the endpoint of interest for a range of botanicals. VERA correctly labelled 70% of the botanicals, indicating the most similar substances and the main features associated with carcinogenicity.     

密度泛函理论研究黄曲霉素B1的表面增强拉曼散射效应

用密度泛函理论B3LYP方法和6—311G（d,p）／Lan12DZ优化得到黄曲霉素B1（AFBI）分子及其复合物AFB1-Ag的稳定结构,并计算了复合物的表面增强拉曼光谱和预共振拉曼光谱．结果表明,AFB1分子的拉曼光谱很大程度依赖于吸附位点以及入射光的激发波长．与分子的常规拉曼光谱相比,复合物表面增强拉曼光谱中C=O伸缩振动模的增强因子约为10^2—10^3,是由于复合物的极化率增强而导致的静态化学增强,并分析了振动模式的振动方向与其拉曼强度的关系．选择复合物最大吸收峰附近激发光266和482nm以及远离共振吸收波长785和1064nm作为入射光,计算得到不同入射光激发下复合物的预共振拉曼光谱．结果表明其增强因子最大达N100量级,主要是由电荷转移产生的共振增强引起的．     

Spectrophotomeric Assay of Aflatoxin B1 Using Acetylcholinesterase Immobilized on Standard Microplates

Aflatoxins are a group of hepatotoxic secondary metabolites produced by molds Aspergillus. During inappropriate storage or processing of food and beverages, aflatoxins can be distributed and become a serious health problem. Disparate analytical techniques were prepared for the assay in the past. Here, a method based on inhibition of enzyme acetylcholinesterase (AChE) is performed allowing fast and simple prove of aflatoxins presence. For the assay purposes, AChE was immobilized on standard polystyrene microplates using gelatin as a stabilizing substance. Both free and immobilized AChEs were used and compared one to each other. Immobilization protocol was optimized and interference of organic solvents such as methanol was tested. For the immobilized AChE, limit of detection 2.75 ppb for aflatoxin B1 was received. The immobilized AChE kept full activity at least one month. Suitability of the assay for a practical performance is considered.     

Research Progress on Fumonisin B1 Contamination and Toxicity: A Review

Fumonisin B1 (FB1), belonging to the member of fumonisins, is one of the most toxic mycotoxins produced mainly by Fusarium proliferatum and Fusarium verticillioide. FB1 has caused extensive contamination worldwide, mainly in corn, rice, wheat, and their products, while it also poses a health risk and is toxic to animals and human. It has been shown to cause oxidative stress, endoplasmic reticulum stress, cellular autophagy, and apoptosis. This review focuses on the current stage of FB1 contamination, its toxic effects of acute toxicity, immunotoxicity, organ toxicity, and reproductive toxicity on animals and humans. The potential toxic mechanisms of FB1 are discussed. One of the main aims of the work is to provide a reliable reference strategy for understanding the occurrence and toxicity of FB1.     

Use of MALDI-TOF MS to Discriminate between Aflatoxin B1-Producing and Non-Producing Strains of Aspergillus flavus

Aflatoxin B₁ (AFB₁) is one of the most toxic mycotoxins. One of the producers of AFB₁ is Aspergillus flavus. Therefore, its rapid identification plays a key role in various sectors of the food and feed industry. MALDI-TOF mass spectrometry is one of the fastest and most accurate methods today. Therefore, the aim of this research was to develop the rapid identification of producing and non-producing strains of A. flavus based on the entire mass spectrum. To accomplish the main goal a different confirmatory MALDI-TOF MS and TLC procedures such as direct AFB₁ identification by scraping from TLC plates, A. flavus mycelium, nutrient media around A. flavus growth, and finally direct AFB₁ identification from infected wheat and barley grains had to be conducted. In this experiment, MALDI-TOF mass spectrometry with various modifications was the main supporting technology. All confirmatory methods confirmed the presence of AFB₁ in the samples of aflatoxin-producing strains of A. flavus and vice versa; AFB₁ was not detected in the case of non-producing strains. Entire mass spectra (from 2 to 20 kDa) of aflatoxin-producing and non-producing A. flavus strains were collected, statistically analyzed and clustered. An in-depth analysis of the obtained entire mass spectra showed differences between AFB₁-producing and non-producing strains of A. flavus. Statistical and cluster analysis divided AFB₁-producing and non-producing strains of A. flavus into two monasteries. The results indicate that it is possible to distinguish between AFB₁ producers and non-producers by comparing the entire mass spectra using MALDI-TOF MS. Finally, we demonstrated that if there are established local AFB₁-producing and non-producing strains of A. flavus, the entire mass spectrum database identification of aflatoxigenic A. flavus strains can be even faster and cheaper, without the need to identify the toxin itself.     

Combined biocompatible medium with molecularly imprinted polymers for determination of aflatoxins B1 in real sample

A kind of molecularly imprinted polymers modified with biocompatible medium was prepared by suspension polymerization. The obtained hybrid materials were used as the adsorbents for the solid‐phase extraction of aflatoxins B1 in real samples. A structural analog of the target, 6‐methyl‐4‐phenylchroman‐2‐one was used as the pseudo‐template, owing to their lower toxicity and cheaper price compared with aflatoxins B1; and methacrylic acid and glycidyl methacrylate were used as the co‐monomers. Scanning electron microscopy and size distribution analysis were used to characterize the obtained polymers. The extraction parameters were optimized to achieve the desired extraction performance. The polymer solid‐phase extraction coupled with high‐performance liquid chromatography was successfully applied to determine aflatoxins B1 from soy sauce without the process of protein removal. Under the optimum extraction conditions, the detection results of aflatoxins B1 in lab‐made column in soy sauce samples was carried out, with a recovery rate of 96%. The established method presented a linear range from 10 to 1000 ng/mL with the coefficient of determination of 0.9994 and the limit of detection of 0.05 ng/mL. Likewise, the inherent selectivity of lab‐made column towards aflatoxins B1, Ochratoxin A, and Zearalenone was demonstrated.     

Novel Enzyme-Linked Aptamer Assay for the Determination of Aflatoxin B1 in Peanuts

Abstract

A novel enzyme-linked aptamer assay is reported for the determination of aflatoxin B₁ (AFB₁). AFB₁ can competitively bind with the immobilized biotin-aptamer and release biotin complementary DNA, leading to the gradual fading of the detection system color with increasing of AFB₁ concentration. In the absence of AFB₁, the biotinylated complementary DNA is not be released from the fixed aptamer. Therefore, the enzyme reaction occurs in the detection system. Under the optimized experimental conditions, the proposed method possessed a wide linear range for AFB₁ from 1 to 80?ng/mL (R² of 0.990) with a low detection limit of 0.36?ng/mL. The method was then applied to detect uncontaminated peanuts fortified with different concentrations of AFB₁. The recovery values were from 82.60% to 94.43%, which indicated the proposed method may be used to detect AFB₁ in food and has potential for the development of test kits.     

Electrochemical Immunosensor Made with Zein-based Nanofibers for On-site Detection of Aflatoxin B1

A disposable electrochemical immunosensor for on-site detection of aflatoxin B1(AFB1), one of the most toxic mycotoxins in agri-food products, was fabricated through a low-cost cut-printing method and then modified with zein/polypyrrole(PPy) electrospun nanofibers onto which anti-AFB1 monoclonal antibodies were immobilized covalently. Fabrication was possible with an innovative and simple approach to adsorb nanofibers onto the working electrode during electrospinning. Electrochemical impedance spectroscopy was employed as the principle of detection, and the data collected with a portable potentiostat were treated with information visualization techniques. The nanostructured immunosensor showed a high sensitivity for AFB1 with a linear detection range from 0.25 to 10 ng mL⁻¹ and a theoretical limit of detection of 0.092 ng mL⁻¹, which is adequate to detect AFB1 in food, according to regulatory agencies.     

光谱法研究黄曲霉毒素B1与人血清白蛋白的结合反应

黄曲霉毒素B1(AFB1)是目前发现的致癌能力最强的真菌毒素,严重危害人畜健康.人血清白蛋白(Human serum albumin,HSA)在结合、运输内源性和外源性等小分子物质方面具有重要的生理功能.研究AFB1与HSA的相互作用机理和作用过程,在分子毒理学上具有重要意义.本研究模拟在人体血液pH条件(pH7.4,离子强度0.1 mol/L),通过荧光淬灭、3D荧光法和圆二色谱(Circular dichroism,CD)等光谱方法研究AFB1与人血清蛋白的相互作用.结果表明,AFB1与HSA的内源荧光淬灭属于静态淬灭,AFB1-HSA在298,303,308和313 K4个温度条件下,结合常数均为104数量级,结合位点都约为l.根据Van't Hoff方程,AFB1-HSA体系是熵增焓减的自发过程,分子间主要作用力为疏水作用和氢键.基于F(o)rster's能量转移,得知AFB1与HSA结合距离为3.31 nm.竞争结合实验表明,AFB1结合在HSA的siteI位点上,靠近色氨酸Trp-214.通过3D荧光分析,AFB1的结合作用导致了HSA氨基酸残基微环境和二级构象发生变化.圆二色谱的分析结果表明,二者的结合使得HSA的α-螺旋含量增加.