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。     

基于非标记适配体结构变化荧光法检测黄曲霉毒素B1

构建了一种基于非标记适配体结构变化荧光检测黄曲霉毒素B1(AFB1)的方法。无AFB1时,一条非标记的AFB1适配体同时与2条短互补DNA链杂交,形成DNA双链结构,导致标记于其中一条互补DNA的3’端的荧光素（FAM）与标记于另一条互补DNA的5’端的淬灭剂（BHQ1）相邻近,发生荧光共振能量转移,FAM荧光被BHQ1淬灭。AFB1存在时,适配体与AFB1结合,而不与互补DNA发生杂交。此时,FAM与BHQ1距离较远,FAM荧光不能被淬灭。通过测量体系荧光强度变化可定量检测AFB1。方法检出限0.2 nmol/L,定量检测范围1.0 nmol/L～4.0μmol/L。该方法无需共价标记适配体,操作简便,特异性好,能够用于检测复杂基质样品中的AFB1。     

基于Cu-TPA的电化学生物传感器对黄曲霉毒素B1的检测

利用对苯二甲酸铜(Cu-TPA)能产生强的电化学信号设计了一种灵敏的电化学生物传感器, 并将其用于测定黄曲霉毒素B1(AFB1). 信号探针中的Cu-TPA含有可产生电化学信号的Cu(Ⅱ), 当加入一定量的AFB1后, AFB1与探针中特定的适配体结合, 使信号探针脱落, 电化学信号降低. 根据电化学信号值的变化实现了对AFB1的检测. 在最佳条件下, 该传感器的检出限为4.2×10 ^-6 ng/mL(S/N=3), 线性范围为10 ^-5~10 ng/mL. 将该传感器用于啤酒中AFB1的检测, 回收率为95%~106%.     

基于纳米多孔硅烷薄膜的适体电化学传感器用于黄曲霉毒素B1的检测

构建了一个基于纳米多孔硅烷薄膜的适体电化学传感器,用于中草药中的黄曲霉毒素B1(AFB1)的灵敏检测。以溶胶-凝胶溶液电沉积再洗脱的方法在活化的玻碳电极表面制备了孔隙均匀,结构稳定的纳米多孔硅烷薄膜,并在此基础上沉积了金纳米粒子,为巯基修饰的适配体提供了大量的活性位点。利用AFB1与适配体的特异性结合能力,将适配体上吸附的信号分子亚甲基蓝（MB）与目标物AFB1形成竞争关系,以此来定量检测AFB1,线性范围在0.01～75μg/L,检出限为5.6 ng/L。方法应用于3种中草药（当归、黄芪、党参）的加标回收检测,结果满意。本传感器的构建为检测AFB1提供了一个新的思路。     

核酸适配体传感器在黄曲霉毒素B1检测中应用研究进展北大核心CSCD

核酸适配体作为一种新型识别分子,具有亲和力高、稳定性强、制备成本低、特异性强等优点,但其自身不具有信号转换功能,它与靶标分子特异性结合过程,不可产生被检测的物理化学信号。因此,需将核酸适配体与靶标分子特异性识别结合过程转为易于被检测的物理化学信号变化的过程。根据信号转换方式的不同,可将适配体生物传感器分为荧光适配体传感器、比色适配体传感器、电化学适配体传感器和表面拉曼散射适配体传感器。本文对基于以上4种检测信号的核酸适配体生物传感器在黄曲霉毒素(AFB1)检测方面的应用进行综述,并概述该类传感器应用前景和当前面临的挑战。     

基于磁珠-适配体的高效液相色谱-串联质谱法检测食品中的黄曲霉毒素B1

本文构建了特异性识别黄曲霉毒素B₁(AFB₁)的磁珠-适配体,并与高效液相色谱-串联质谱联用(LC-MS/MS),建立食品中AFB₁的定量检测方法。 利用碳二亚胺盐酸盐(EDC)活化法,将羧基磁珠进行活化。 活化后的羧基磁珠与5'端氨基修饰的适配体进行孵育结合,通过酰胺反应将适配体共价连接在羧基磁珠表面,固定在磁珠表面的适配体作为捕捉探针将样品提取液中的AFB₁分离,通过LC-MS/MS对AFB₁进行定性和定量分析。 检测结果表明:AFB₁在浓度0.25～25 ng/mL呈良好的线性关系,相关系数R²=0.999,定量检出限为0.25 ng/mL,回收率达到80.3%～92.5%,相对标准偏差(RSD)低于8%。 该方法操作简单、快速便捷、可痕量地检测AFB₁,所制备的磁珠-适配体可重复利用,为定量检测AFB₁提供了另一种技术支持。     

纳米金-适配体比色传感法检测玉米油中黄曲霉毒素B1

黄曲霉毒素B₁(Aflatoxin B₁, AFB₁)是真菌或霉菌产生的有毒代谢物,通过污染农作物和食品从而对人体健康造成严重威胁。本研究建立了纳米金(AuNPs)-适配体比色传感法,采用不同序列长度的AFB₁适配体检测玉米油中AFB₁。通过优化适配体浓度、NaCl浓度和体系反应温度,进一步提高体系检测灵敏度。在最佳条件下,50(B50)和80(A80)个碱基长度的适配体建立的AuNPs-适配体比色传感法的检出限分别为13.55和10.56 ng/mL,线性范围均为20～1000 ng/mL,且选择性良好。基于B50和A80适配体比色传感法检测玉米油中AFB₁的加标回收率分别为102.4%～104.9%和98.1%～109.1%。结果表明,基于B50和A80建立的AuNPs比色传感法均可用于玉米油中AFB₁特异性鉴定。本研究为现场快速检测和高效筛查食品中AFB₁污染提供了技术支持,为开发针对其它靶标的适配体传感检...     

快速检测中药材中黄曲霉毒素B1的电化学生物传感器

结合新型纳米传感膜材料,以黄曲霉毒素Bl(AFBl)单克隆抗体为生物识别元件,通过考察AFB1与抗体之间的相互作用对测试底液中[Fe(CN)6]3-/[Fe(CN)6]4-([Fe(CN)6]3-/4-)氧化还原体系的影响,构建了一种可用于中药材中AFB1快速检测的电化学生物传感器.在最佳条件下,对AFB1浓度的线性响...     

基于酶辅助靶标循环的适体传感器灵敏检测中药中黄曲霉毒素B1

该文基于酶辅助靶标循环信号放大策略构建了用于黄曲霉毒素B1(AFB1)高灵敏检测的化学发光适体传感器。以G-四链体/氯化血红素DNA酶为信号分子设计了免标记的适体探针H1-S1和发夹探针H2。适体探针结合目标AFB1,在核酸外切酶I辅助下,触发靶标循环反应产生发夹H1。发夹H1与H2杂交,释放出完整的G-四链体序列,并进一步与氯化血红素结合形成G-四链体/氯化血红素DNA酶。DNA酶通过催化氧化鲁米诺-H2O2化学发光体系产生化学发光信号,实现AFB1的放大检测。在最优实验条件下,化学发光强度与AFB1质量浓度的对数在0.001~100 ng/mL范围内呈良好的线性关系,相关系数(r2)为0.9955,检出限为0.93 pg/mL,回收率为93.7%~107%。该适体传感器操作简单、灵敏度高、特异性好,在黄曲霉毒素污染检测方面具有良好的应用前景。     

一种超灵敏检测镉离子的核酸适配体电化学传感器

设计了一种基于核酸适配体检测镉离子(Cd²⁺)的电化学生物传感器,将适配体互补链(CDNA)通过AuS键自组装于金电极表面,并与适配体杂交结合形成双链DNA。由于适配体对Cd²⁺有特异性结合能力,加入Cd²⁺后,与互补链竞争结合适配体,使修饰二茂铁基团的适配体从金电极表面脱落,二茂铁的电化学信号显著减小。采用方波伏安法(SWV)进行检测,本传感器对Cd²⁺的线性检测范围为1.0 nmol/L～10.0μmol/L,检出限为65.1 pmol/L,线性方程为ΔI=0.2872+0.2327lgC(R²=0.9972), 10 s内即可完成检测。实际江水样品中Cd²⁺的检测结果与石墨炉原子吸收光谱法的检测结果一致,加标回收率为97.1%～99.5%。本方法灵敏度高、检测速度快、特异性强,在镉环境污染监测方面具有良好的应用前景。     

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.     

A Novel Electrochemical Method for the Sensitive Determination of Aflatoxin B1 Using a Bivalent Binding Aptamer-cDNA Structure

In this study, a simple electrochemical sensing platform with the employment of a bivalent binding aptamer-cDNA probe (BBA-cDNA) structure is constructed for the detection of aflatoxin B₁ (AFB₁) as a mycotoxin. The BBA-cDNA structure is composed of two strands of aptamer (Apts) and their complementary strand (CS). Using a simple but accurate design, the presented measurement approach showed enhanced sensitivity and selectivity for AFB₁ detection with a LOD of 0.1 ng/mL. The approach presented in this study can be applied to the development of biosensors for the measurement of various toxins by substituting the proper aptamers and complementary strands.     

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。     

Fe3O4 Nanorods-RGO-ionic Liquid Nanocomposite Based Electrochemical Sensor for Aflatoxin B1 in Ground Paprika

A novel and sensitive method for the determination of aflatoxin B1 (AFA−B1) in ground paprika using a methyltrioctylammonium chloride ionic liquid (IL), iron oxide nanorods (Fe₃O₄ nanorods) and reduced graphene oxide (RGO) fabricated glassy carbon electrode (GCE) was developed. The synthesized nanoparticles, nanocomposites and modified electrode surfaces were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), thermogravimetric analysis (TGA/DSC) and x-ray diffraction (XRD) analyses. Moreover, the electrochemical performance of the developed sensor was determined by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The obtained results demonstrate that the sensitivity of AFA−B1 is significantly enhanced on RGO-Fe₃O₄ nanorods-IL-GCE in comparison with bare GCE, RGO-GCE and RGO-Fe₃O₄ nanorods-GCE. The redox peak currents of AFA−B1 exhibited good linear relationship with its concentration in the range from 0.02 to 0.33 ng mL⁻¹ with detection limit of (LOD) 0.03 ng mL⁻¹ and limit of quantification (LOQ) 0.36 ng mL⁻¹ respectively (S/N=3). In addition, the fabricated electrode showed good stability and reproducibility. The proposed technique was effectively applied to identify the AFA−B1 in real ground paprika samples with acceptable results.     

Electrochemical Aptasensor Based on Poly(Neutral Red) and Carboxylated Pillar[5]arene for Sensitive Determination of Aflatoxin M1

Aptasensor for highly sensitive determination of aflatoxin M1 (AFM1) was developed on the base of glassy carbon electrode (GCE) covered with polymeric Neutral red (NR) dye obtained by electropolymerization in the presence of polycarboxylated pillar[5]arene derivative. Aptamer against AFM1 and NR label were then covalently linked to the carboxylic groups of the carrier by carbodiimide binding. At presence of AFM1 the cathodic peak current related to the NR conversion decreases. AFM1 induced also an increase of the charge transfer resistance measured by electrochemical impedance spectroscopy. In optimal conditions, this make it possible to determine from 5 to 120 ng/L AFM1 in standard solutions with limit of detection (LOD) of 0.5 ng/L. The aptasensor was validated on the spiked samples of cow and sheep milk as well as in kefir after their methanol dilution. Reliable detection of the 40–160 ng/kg of mycotoxins was reached. This is below limited threshold value (50 μg/kg) established in EC.     

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.     

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.     

Disposable Electrochemical Aptasensor for Ultrasensitive Determination of Aflatoxin B1 Using Copper Nanoparticles as Probes

An electrochemical aptasensor for aflatoxin B1 (AFB1) detection was constructed based on the copper nanoparticles (CuNPs) and gold nanoflowers modified screen-printed carbon electrodes as electrochemical probes and substrates, respectively. In the range of 100 aM to 100 pM, a good linear relationship between oxidation peak current of CuNPs and concentration of AFB1 was obtained. The high sensitivity could be ascribed to the amplified electrochemical signals by CuNPs. The high affinity of AFB1 with aptamer endowed its high selectivity. The above advantages and disposable traits made this aptasensor as an ideal platform for evaluation of AFB1 level in food samples.