果汁中脂环酸芽孢杆菌磁性荧光量子点快速检测理论方法

Alicyclobacillus spp. are one of the most important quality and safety factors of fruit juice and have become a major issue in the fruit juice processing industry. The development of rapid and accurate methods for the detection and identifying of Alicyclobacillus spp. in fruit juice products has become a focus of investigation. In this project, magnetic-fluorescent quantum dots will be prepared based on the magnetic nanoparticles and fluorescence quantum dots, the coupling way and binding sites are assisted by analysis of the molecular composition and chemical structure. The immobilization methods and conditions of specific anti-Alicyclobacillus antibodied onto magnetic nanoparticles will be optimized, the functional magnetic composite materials, which can be used for the immunomagnetic separation and detection of Alicyclobacillus spp., will be generated. Meanwhile, the molecular mechanism for the specific anti-Alicyclobacillus antibodies binding to magnetic-fluorescent quantum dots are revealed by the analysis of structure compositions and biological properties. The most suitable function range, methods and specificity of functional magnetic composite materials for the magnetic separation of Alicyclobacillus spp. are evaluated, and the detection limit and accuracy are comfirmed. Based on the linear equation and least square method, the model will be establised for rapid detection and quantitative analysis of Alicyclobacillus spp. in fruit juice. Finally, the theoretical and methodology systems for magnetic separation and rapid detection of Alicyclobacillus spp. from fruit juice by magnetic-fluorescent quantum dots will be established. This work is believed to be of both academic interest and practical importance for monitoring Alicyclobacillus spp. in fruit juice and other beverage industry.

脂环酸芽孢杆菌是果汁产业中关键的质量安全因子，研究建立高效、快速的检测方法，及时识别、发现与快速检测脂环酸芽孢杆菌的污染，一直是中国果汁国际化发展的技术瓶颈之一。本项目通过磁性纳米微球偶联荧光量子点，制备获得性能优良的磁性荧光量子点复合材料，对其分子组成及化学结构进行解析，揭示磁性纳米微球和荧光量子点耦合的作用方式与结合位点；优化建立基于磁性荧光量子点复合材料的脂环酸芽孢杆菌多克隆抗体偶联固定化方法，制备获得兼具磁性分离及荧光检测特性的功能化磁性荧光量子点复合材料，分析评价其结构组成和生物性能，明确多克隆抗体固定化的作用位点与分子机制；确定功能化磁性荧光量子点复合材料对脂环酸芽孢杆菌磁性分离的特异性、最佳作用方式与范围，通过线性方程和最小二乘法优化建立脂环酸芽孢杆菌快速检测和定量分析模型，明确快速检测方法的灵敏度、准确性，系统构建果汁中脂环酸芽孢杆菌磁性荧光量子点快速检测技术方法体系。

脂环酸芽孢杆菌是果汁产业中关键的质量安全因子，研究建立高效、快速的检测方法，及时识别、发现与快速检测脂环酸芽孢杆菌的污染，一直是中国果汁国际化发展的技术瓶颈之一。本研究主要以磁性纳米材料、荧光量子点和多克隆抗体为基础，构建了针对苹果汁中脂环酸芽孢杆菌的快速分离与荧光量子点检测方法体系。主要获得如下结果：（1）优化合成了核壳型CdTe/CdS量子点，采用荧光发射光谱及紫外可见光谱等对量子点的光学性能进行了评价，探究了量子点的稳定性。研究结果表明：在80℃回流，成核40 min，包壳40 min，pH=11，n（Cd2+)：n（Te2-）：n（TGA）=1：0.2：7的条件下，制备的量子点具有较优良的性能。稳定性研究结果表明制备的CdTe/CdS量子点随照射时间的延长，光致发光强度明显增强，量子产率也明显增加。研究建立的量子点制备方法为检测方法的建立提供了技术支撑。（2）以脂环酸芽孢杆菌标准菌株为抗原免疫新西兰大耳白兔制备多克隆抗体，并对其性能进行了评价，结果表明：免疫的三只新西兰大耳白兔获得的抗体浓度分别为11.52 mg/ml、9.86 mg/ml及9.91 mg/ml；SDS-PAGE电泳显示抗体相对分子量为150KD；抗体的效价为80000。特异性评价结果表明：制备获得的多克隆抗体对脂环酸芽孢杆菌具有非常好的特异性。（3）以材料学、免疫学及微生物学为基础，构建获得了兼具多克隆抗体特异识别功能及磁性荧光量子点检测性能的功能化复合材料，以此优化建立了果汁中脂环酸芽孢杆菌的快速富集与检测方法体系。研究结果表明：通过硫醇化学反应可以实现MNPs- SiO2-SH纳米粒子与功能化的CdTe / CdS量子点有效结合，制备的MNPs-QD纳米复合材料具有良好的磁响应特性和荧光特性；通过酯化反应可以实现脂环酸芽孢杆菌多克隆抗体的固定化，其固定化量为119.62μg/mg，吸附反应60min；吸附过程符合Langmuir模型和准二级动力学方程；建立的检测方法对脂环酸芽孢杆菌的检测限为104 CFU/mL，检测过程90 min。结果表明，以构建的MNPs-QDs-抗体复合物实现了苹果汁中目标物的高效捕获和检测，为果蔬产业中目标物的检测识别提供了新的方法思路。