【作者】 徐霞；

【导师】 应义斌； 李延斌；

【作者基本信息】 浙江大学 ， 生物系统工程， 2013， 博士

【摘要】 真菌毒素在全球范围内威胁农作物和农产品的安全,造成的直接或间接经济损失严重,同时经由污染的农产品和食品可对人体健康产生严重危害甚至死亡,其中尤以黄曲霉毒素和赭曲霉毒素毒性最大、污染最为严重。传统检测方法如薄层色谱、高效液相色谱以及酶联免疫吸附在检测时间、设备成本或操作等方面存在局限。基于金属纳米粒子的光学生物传感方法具有快速、简单的优势,是一种潜在的解决手段。其中金纳米棒这种各向异性的一维纳米材料,因独特的光学特性,在近年来备受关注。本课题以花生、谷物等农产品中常见的真菌毒素黄曲霉毒素B1(aflatoxin B1,AFB1)和赭曲霉毒素A (ochratoxin A, OTA)为检测对象,以特异性单克隆抗体和适配体(aptamer)为生物识别元件,分别建立了基于金纳米棒局域等离子体共振(localized surface plasmon resonance, LSPR)吸收光谱和动态光散射(dynamic light scattering, DLS)的生物传感方法和定量检测模型。首次探索了基于金纳米棒的多种生物传感方式对小分子真菌毒素的简单、快速检测,为农产品中真菌毒素的现场测定提供了一种新思路。本文的主要内容和研究结果如下：(1)分析和研究了贯穿整个课题的纳米材料-金纳米棒的物理和化学特性。基于透射电子显微镜、紫外-可见-近红外吸收光谱和拉曼光谱、动态光散射、电感耦合等离子体质谱等手段对实验室制备的金纳米棒的尺寸、水合粒径、浓度和光谱特性等进行了表征。稳定性研究结果显示：金纳米棒在不同pH(2-13范围内)的CTAB溶液中均表现出较高的稳定性；在高浓度CTAB体系中,金纳米棒抗离子强度的稳定性较强；金纳米棒与浓度在0-10mM或大于100mM的磷酸盐缓冲液混合后比较稳定。金纳米棒特性分析是本课题研究的基础,也是金纳米棒分析和应用的理论依据。(2)以甲胎蛋白(a-fetoprotein, AFP)为模型,尝试和比较了基于末端和全表面抗体修饰的金纳米棒的不同免疫传感应用。1)建立了基于末端修饰的金纳米棒的直接免疫传感方法：通过直观的金纳米棒LSPR波长的偏移来识别金纳米棒端面AFP结合后周围环境折射率的变化,此方法线性检测范围相对较窄,对设备要求较高；通过吸收强度变化来识别金纳米棒末端修饰的AFP分子,可建立定量模型,但检测性能不理想。2)建立了基于全表面抗体修饰的金纳米棒直接聚集的免疫传感方法：金纳米棒纵向LSPR偏移与AFP标准溶液(0.25-4.0nM)线性定量模型的检测限和定量限分别为0.18和0.60nM；金纳米棒LSPR吸收强度与在PBS缓冲液中AFP标准溶液(0.25-4.0nM)定量模型的检测限和定量限分别为0.04和0.14nM,选择性试验中干扰蛋白所引起的吸收强度信号与单纯AFP的信号相比变化小于5.3%。结果表明,金纳米棒直接聚集导致的等离子体共振耦合信号增强为小分子真菌毒素的定量检测提供了可行性。(3)建立了一种基于金纳米棒竞争分散方式的免标记、简单一步快速检测的免疫传感方法,并将其用于农产品中黄曲霉毒素B1的测定。功能化的GNR-AFB1-BSA偶联物与AFB1特异性单克隆抗体混合后发生不可逆的聚集,导致金纳米棒吸收强度的下降以及水合粒径的显著增大；当样本中游离AFB1分子存在时,可与GNR-AFB1-BSA偶联物竞争结合到特异性抗体上,使得金纳米棒在溶液中呈分散的状态。所建立传感方法可有效避免复杂环境应用时因非特异性聚集而产生的假信号。金纳米棒的吸收强度被用作传感信号,同时液体样本中金纳米棒平均水合粒径的测量也被用作传感的方法。研究了不同抗体浓度对金纳米棒聚集程度和AFB1定量检测结果的影响：当抗体浓度为15μg/mL时,所建立吸收强度与AFB1浓度的线性定量模型的检测限为0.035ng/mL,但检测范围较窄(0.1-1.0ng/mL);当抗体浓度为25μg/mL时,线性定量模型的检测限为0.16ng/mL,线性检测范围为0.5-20ng/mL,满足欧盟标准的测定需求。利用其他三种黄曲霉毒素和四种农产品中常见真菌毒素对所建立的免疫传感器的选择性进行评估。并将建立的传感方法应用于真实花生样本的分析,经计算加标回收率在94.2%-117.3%范围内。结果表明,基于金纳米棒竞争分散方式的免疫传感方法可快速、简单定量检测AFB1,同时对真实样本的测定具有良好的准确度和可信度。(4)探索了基于金纳米棒肩并肩与头碰头组装结构的适配体传感器(aptasensor)用于赭曲霉毒素A的快速、灵敏检测。修饰在金纳米棒全表面和两个端面的适配体与互补脱氧核糖核苷酸(deoxyribonucleic acid,DNA),在一定条件下可发生核酸杂交(hybridization),诱导金纳米棒发生肩并肩或头碰头的组装,形成链状结构；当溶液中存在目标配体OTA小分子时,适配体构象发生变化,形成G-四聚体结构将OTA埋在其中,此时适配体无法与互补DNA杂交,金纳米棒组装结构减少,可实现真菌毒素OTA的检测。本文构建并表征了金纳米棒肩并肩和头碰头两种组装结构,其中肩并肩组装的金纳米棒的等离子体耦合更为显著,可进一步用于OTA的测定。将两种全表面修饰的金纳米棒探针与不同浓度的OTA标准溶液(0-100ng/mL)混合,结果显示：随着OTA浓度增大,金纳米棒纵向LSPR吸收强度逐渐增大。由此根据706nm波长处吸收强度可以建立OTA定量检测的方法。同时利用农产品中其他五种常见的真菌毒素评估基于金纳米棒肩并肩组装结构的适配体传感器的特异性。结果表明,基于金纳米棒肩并肩组装结构的光学适配体传感方法具有高灵敏、高特异性定量测定农产品中OTA的潜力。(5)搭建了真菌毒素检测用便携式快速检测平台,并将其应用于花生样本中AFB1含量的测定。基于该平台,应用金纳米棒竞争分散的传感方法建立AFB1标准溶液浓度与吸收强度之间的关系。在0-5.0ng/mL浓度范围时,金纳米棒吸收强度(709nm)与AFB1浓度呈线性关系,检测限为0.066ng/mL,线性检测范围为0.22-5.0ng/mL。利用该平台可测定当地农贸市场购买的新鲜花生和实验室保存8个月的花生样本中的AFB1含量,加标回收率理想分别为97.6%和95.7%,检测结果与高效液相色谱标准方法的测定结果基本一致。结果表明,基于此平台的真菌毒素检测用新型光学纳米生物传感器具有测量结果准确可信、分析成本低、快速、体积小便于携带等特点,适用于真菌毒素的现场检测。更多还原

【Abstract】 The damage of crops and agro-products caused by mycotoxins induces serious economic losses directly and indirectly on a global scale. Many mycotoxins can cause serious harm to human health and even death in people through mycotoxin-contaminated food, especially aflatoxins and ochratoxins which are most toxic and polluted. Traditional detection methods such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and enzyme-linked immuno sorbent assay (ELISA) are sensitive; however, more rapid, simple, and cost-effective approaches are still requested by the food industry. More recently optical biosensors coupled with metal nanoparticles provides a promising potential approach for the purpose, which is perhaps one of the most powerful and simple nanosensing methods available. Wherein gold nanorods (GNRs) as one kind of anisotropy one-dimensional materials, have received much attention in the past decade due to their unique optical properties.In this study, aflatoxin B1(AFB1) and ochratoxin A (OTA) in common agro-products like peanuts and grain were selected as detection target of interest. Biosensing methods and quantitative detection models based on localized surface plasmon resonance (LSPR) absorption spectra and dynamic light scattering (DLS) of GNRs have been established, with biological recognition elements of specific monoclonal antibody and aptamer. It is the first time to explore the available biosensing approaches based on GNRs for rapid and simple discrimination of mycotoxins, providing a new platform for on-site testing of mycotoxin contamination in common agro-products.The main contents and results are summarized as follows:(1) The physical and chemical properties of GNRs were studied initially. Various techniques including transmission electron microscopy (TEM), UV-vis-NIR absorption spectra, dynamic light scattering and inductively coupled plasma mass spectrometry (ICP-MS) were used for size, hydrodynamic diameter, concentration and optical property characterization of GNRs. The results of stability study showed that:GNRs could keep stable in CTAB solution of different pH environment (pH value from2to13); higher stability of GNRs could been observed under strong ionic strength conditions at a higher CTAB concentration, and GNRs got irreversible aggregation while mixing with NaCl solution concentration greater than160mM; GNRs could keep stable in biological analyzing buffer of phosphate buffered saline (PBS) with concentration in the range of0-10mM or greater than100mM. It provided theoretical basis for analysis and application based on GNRs.(2) Different biosensing method based on end and satellite antibody modified GNRs were established and compared, using alpha-fetoprotein (AFP) as a model analyte which is one of the major markers for hepatocellular tumors. Direct immune sensing method with end modified GNRs was investigated. Change in refractive index of surrounding environment after AFP binding was recognized according to shift of longitudinal LSPR wavelength, however the linear detection range of this method was narrow (0.25-1.0nM) with high requirement of equipment in resolution and signal to noise ratio. Absorption intensity of normalized spectra could also been used for AFP sensing (0.25-14.3nM), however the detection performance was not ideal. For immune sensing approach with satellite modified GNRs, dramatic aggregation of GNRs was induced by the interaction between target proteins and capture antibodies. Associated wavelength red-shifts and intensity decreases in the absorption peak of GNRs were observed with increasing concentrations of target AFP. It was illustrated that this method can be used reliably to detect AFP protein in PBS solution at concentrations of picomolar level with a wide quantification range from0.25to4.0nM. The limit of detection and quantification based on wavelength shift was0.18and0.60nM, and based on absorption intensity was0.04and0.14nM, respectively. The complication of interfering proteins in the sample showed negligible effects on the detection results. It is expected that plamon coupling based signal enhancement caused by GNR aggregation would be applicable for discrimination of mycotoxins in agro-products at low concentrations.(3) A promising one-step and label-free optical biosensor was illustrated for determination of AFB1that is most commonly found in foods and highly dangerous even at very low concentrations. In this research, GNRs were employed as a sensing platform, which showed high stability under high ionic strength conditions without addition of any stabilizing agent. GNR-AFB1-BSA (bovine serum albumin) conjugates aggregated after mixing with free antibodies, resulting in significant changes in absorption intensity. At the same time the existence of AFB1molecules in samples caused dispersion of nanorods, as a result of competitive immune-reaction with antibodies. By taking advantages of the competitive dispersion of GNRs, the developed method could effectively reduce false results caused by undesirable aggregation, which is a big problem for spherical gold nanoparticles. Absorption intensity of UV-vis spectra served as the sensing indicator, with dynamic light scattering measurement as another sensing tool. While the concentration of AFB1antibody was15μg/mL, the biosensing system could detect AFB1standard PBS solution linearly from0.1to1.0ng/mL, with a low limit of detection (LOD) of0.035ng/mL. While the concentration of AFB1antibody was25μg/mL, the designed biosensing system could detect AFB1in a linear range from0.5to20ng/mL, with a good correlation coefficient of0.99. And the limit of detection was0.16ng/mL, indicating an excellent sensitivity with absorbance result. The recoveries of the spiked AFB1in real peanut samples ranged from94.2%to117.3%. Therefore the proposed nano-biosensor was demonstrated to be sensitive, selective, and simple, providing a viable alternative for rapid screening of toxins in agriculture products and foods.(4) Rapid and sensitive aptasensors based on GNR side-by-side and end-to-end assemblies were explored for OTA screening. Aptamer and complementary DNA (cDNA) modified on satellite and end faces of GNRs hybridized under certain conditions, resulting in chain-like structure induced by side-by-side and end-to-end assembly of GNRs. The conformation of aptamer changed in the presence of free OTA molecule in solution, forming a G-quartet of OTA aptamer. As a result, the GNR assemblies reduced with the increasing of OTA concentration, which could been used for OTA detection. GNR side-by-side and end-to-end sassemblies were characterized and optimized. Plasmon coupling of side-by-side GNR assembly was observed stronger than that of end-to-end assembly, which was potential for OTA detecton. Thus, satellite-modified GNR-aptamer and GNR-cDNA probes mixed well and reacted with different concentration of OTA standard solution (0-100ng/mL), and the absorption intensity of GNRs at706nm could be employed as signal indicator for quantification of OTA. Other five mycotoxins common in agro-products were detected for evaluation the selectivity of the proposed aptasensor. It was indicated that the optical aptasensor based on GNR side-by-side assemblies could been potential for OTA discrimination with high sensitivity and specificity.(5) A portable and rapid detecting platform for mycotoxin was constructed, and applied for determination of AFB1content in peanut samples. The relationship between AFB1concentration and absorption intensity of UV-vis spectra at709nm was set up using the platform based on competitive dispersion of GNRs. AFB1in standard PBS solution could be detected linearly from0.22to5.0ng/mL, with a low limit of detection of0.066ng/mL. AFBl in fresh and stored peanut samples purchased from local agricultural market were detected, resulting in ideal spiked recoveries of97.6%and95.7%, respectively. Additionally, the measurement results kept consistent with standard method of HPLC results. Therefore it was indicated that the novel optical nano biosensor based on the portable platform was promising for on-site detection of mycotoxins, with advantages of accurate, reliable and fast measurements, cost-effective, small-size and easy to carry.更多还原