基于纳米颗粒的可视化生物芯片检测技术研究

发布时间：2018-04-23 18:01

  本文选题：量子点 + 生物素-酪胺　； 参考：《安徽医科大学》2012年硕士论文

【摘要】：目的荧光检测是生物芯片的常规检测方法。该方法最大的缺点是荧光扫描仪器价格昂贵，而且体积笨重，限制了生物芯片技术在中小型医疗机构以及现场检测中的应用。近年发展起来的一些纳米材料如纳米金颗粒、量子点、纳米银颗粒由于表现出各种独特的光电性质，在分子检测领域引起了广泛关注。本实验的目的是基于纳米颗粒建立高检测灵敏度可视化生物芯片检测技术，使生物芯片技术能够广泛地应用于分子检测。方法通过对现有HRP底物的修饰改造，合成一系列新型HRP底物。利用生物偶联方法制备了一种新的纳米金复合底物，实现了HRP直接催化纳米金颗粒的沉积，评价了该底物用于生物芯片可视化检测的灵敏度,并将纳米金底物用于基因芯片和蛋白芯片的可视化检测。以基因芯片可视化检测为例，确定检测流程、优化检测条件、比较纳米金复合底物T-Au、TSA-GLSS、GLSS检测灵敏度。 量子点是一种新型纳米颗粒，本研究以基因芯片分析技术为平台，将TSA结合量子点标记银染增强技术，建立一种基于量子点标记银染增强可视化检测方法。量子点标记银染增强可视化检测方法：待测靶基因与固定在玻片上的探针杂交，依次加入链霉亲和素标记的辣根过氧化物酶、生物素标记的酪胺以及链霉亲和素标记的量子点，37℃孵育，然后加入银增强试剂显色；最后使用可视化生物芯片扫描仪扫描并记录结果。以牛布鲁氏菌为检测对象,比较TSA-Cy3和TSA-QDS两种检测法的检测灵敏度。 结果纳米金新底物制备条件：T-Au复合底物的制备，是将纳米金的琥珀酰亚胺酯衍生物（Nanogold-NHS）与酪胺在50℃下避光反应1.5h即可生成。所制备的纳米金复合底物T-Au基因芯片可视化法检测流程为：固定在玻片上的探针与待测靶标杂交后依次加入streptavidin-HRP和T-Au的稀释液、温育。加入银增强试剂显色并扫描分析。检测条件为：T-Au稀释比例1:200，，37℃孵育时间25min，银染增强时间4-5min。T-Au与TSA-GLSS检测灵敏度均为103CFU"mL。T-Au检测与TSA-GLSS检测灵敏度相同。 确定了基因芯片量子点标记银染增强可视化检测方法的检测流程，优化了检测条件并考察了检测灵敏度。检测条件为：酪胺-生物素稀释比例1:4000，链酶亲和素标记的量子点稀释比例1:50，37℃孵育时间25-30min，银染增强时间6-7min。TSA-Cy3和TSA-QDS两种检测法检测布鲁氏菌,结果TSA-Cy3和TSA-QDS检测灵敏度均为103CFU/mL。 结论制备了一种纳米金复合底物，实现了HRP直接催化纳米金颗粒的沉积。以所制备的纳米金复合底物基础，建立了T-Au可视化检测方法。该检测方法操作简单，结果可视化，实现了生物芯片高灵敏度可视化检测，可以取代TSA-GLSS可视化检测方法。建立了一种基于量子点标记银染增强的基因芯片的可视化检测方法。本方法具备仪器要求低、结果可视化等优势，为量子点在分子检测领域的应用提供了参考。
[Abstract]:Objective fluorescence detection is a routine method for biochip detection. The biggest drawback of this method is that the fluorescent scanner is expensive and bulky, which limits the application of biochip technology in small and medium-sized medical institutions and field detection. Some nanomaterials, such as gold nanoparticles, quantum dots and silver nanoparticles developed in recent years, have attracted wide attention in the field of molecular detection because of their unique optoelectronic properties. The purpose of this experiment is to establish a high sensitivity visual biochip detection technology based on nanoparticles, so that biochip technology can be widely used in molecular detection. Methods A series of new HRP substrates were synthesized by modifying the existing HRP substrates. A new nano-gold composite substrate was prepared by biological coupling method. The deposition of gold nanoparticles directly catalyzed by HRP was realized. The sensitivity of the substrate for visual detection of biochip was evaluated. The nano-gold substrate was used to visualize the detection of gene chip and protein chip. Taking the visualization detection of gene chip as an example, the detection process was determined and the detection conditions were optimized to compare the sensitivity of T-Au-Au-TSA-GLSSS-GLSS. Quantum dots (QDs) are a new type of nanoparticles. Based on the technology of gene chip analysis, a visual detection method based on QDs was established by combining TSA with QDs. Quantum Dot labeling silver staining enhanced visual detection method: the target gene to be tested was hybridized with the probe fixed on the slide, and the horseradish peroxidase labeled by Streptomycin was added in turn. Biotin labeled tyramine and streptavidin labeled quantum dots were incubated at 37 鈩

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