A Lipobead Based Microarray Platform for the Label-Free Detection of Bacterial Exotoxins

本项目开发一种用于细菌外毒素检测的超小型化微阵列平台。细菌外毒素是许多人类疾病的主要毒力因素。由于它们很容易通过食物和水传播途径传播，对公众健康构成重大威胁，特别是在食物和水源不安全的不发达国家。它们被用作生物恐怖主义的工具是不容忽视的。针对这些病原体的第一道防线是快速检测，但随着病原体数量的激增，这在过去几年中变得更加困难。本项目中使用的微阵列平台专门解决了对快速检测系统的需求，可以从数百种可能性中识别出毒素。微阵列显示了捕获分子的集合，每个分子都选择性地与不同的毒素结合。该阵列使用的捕获分子是天然的膜受体，外毒素在感染细胞之前利用它来瞄准并附着在细胞上。因此，这些受体以高选择性和亲和力结合毒素。受体首先被隔离在支撑磷脂双分子层中，这些磷脂双分子层是围绕十微米大小的聚苯乙烯颗粒形成的。分批制备脂珠，每批显示一个单一的受体，其身份由封装在珠内的发光纳米晶体（量子点或Qds）的发射光谱组成的光谱条形码编码。每个脂粒都有不同的受体和识别条形码，然后通过将它们附着在表面上预先图案的微孔上排列在表面上。这项研究将开发一种荧光检测系统，用于识别毒素与阵列中特定捕获分子的结合，而无需对毒素进行标记或对结合的毒素进行后续分析。无标记系统是基于测量在双分子层中预先标记的供体和受体荧光对之间共振能量转移的变化。与目前通常需要进一步分析步骤的方法相比，这种分析方法将允许更短的筛选时间。小型化的设计还允许在双层中显示不同浓度的受体，这将允许测量结合亲和力。这些额外的信息使毒素的识别更加准确，特别是在两种毒素与同一受体结合的情况下。智力优势：本提案中开发的用于毒素检测的超小型化，无标签微阵列允许从数百种潜在列表中快速筛选分析物样品以检测特定毒素。该设计可用于膜受体的一般显示，以了解药物发现中的结合相互作用，并且用作阵列元件的脂珠也可用于流式细胞术。更广泛的影响：这项研究将支持两名研究生和本科生，他们都将接受界面和生物科学方面的教育。他们的研究还将与城市学院化学工程系的软材料IGERT项目合作，该项目的重点是教育学生在软物质物理学的各个学科。

CBET-0829052MaldarelliThis project develops a ultra-miniaturized microarray platform for the detection of bacterial exotoxins. Bacterial exotoxins are the primary virulence factors for many human diseases. As they are easily carried through food and water borne pathways, they pose a significant threat to public health, especially in underdeveloped countries where food and water sources are not secure. Their use as an instrument of bioterrorism cannot be overlooked. The first line of defense against these pathogens is a rapid detection, but this has become more difficult over the past several years as the number of pathogens has proliferated.The microarray platform used in this project specifically addresses the needs for detection systems which are rapid and can identify a toxin from among hundreds of possibilities. The microarray displays an assembly of capture molecules, each of which selectively binds to a different toxin. The capture molecules used by the array are the natural membrane receptors which the exotoxins use to target and attach to cells before infecting them. As such, these receptors bind the toxins with high selectivity and affinity. The receptors are displayed in the array by first sequestering them in supportedphospholipids bilayers which are formed around polystyrene particles of the order of ten microns in size. Batches of lipobeads are prepared separately, with each batch displaying a single receptor whose identity is encoded by a spectral barcode composed of the emission spectrum of luminescent nanocrystals (quantum dots or Qds)encapsulated in the bead. Lipobeads, each with a different receptor and identifying barcode, are then arrayed on a surface by attaching them to microwells pre-patterned on the surface. This research will develop a fluorescence detection system for identifying the binding of a toxin to a particular capture molecule in the array without having tolabel the toxin or perform subsequent assays on the bound toxin. The label free system is based on measuring the changes in the resonance energy transfer between donor and acceptor fluorescence pairs pre-labeled in the bilayer. This as is assaying will allow a much shorter screening time compared to present approaches which usually require further analytical steps. The miniaturized design also allows for the display of receptors at different concentrations in the bilayers which will allows the binding affinity to be measured. This additional information allows toxins to be identified with greater accuracy, particularly in the case where two bind to the same receptor.Intellectual Merit: The ultra-miniaturized, label-free microarray for toxin detection developed in this proposal allows for the rapid screening of analyte samples for the detection of a particular toxin from a potential list of hundreds. The design can be adopted to the general display of membrane receptors for understanding binding interactions in drug discovery, and the lipobeads used as array elements can alo be used in flow cytometry.Broader Impact: The research will support two graduate students and undergraduates, each of which will be educated in interfacial and biological sciences. Their research will also be in collaboration with the Soft Material IGERT Program in the Department of Chemical Engineering at City College whose focus it is to educate students in the various disciplines of soft matter physics.