Multiplexed GPCR Characterization Using SPR

项目摘要 这个第二阶段STTR项目的目标是开发一种实时无标记的生物传感器， 一次分析96个样本，而当前的分析系统只能分析6个样本。 技术.这个平台最初将被证明与G蛋白偶联受体 （GPCR）和抗体。这两个应用程序的共同点是需要更高的- 吞吐量传感，并展示这些集成系统将说明其 多功能性和潜在的贡献，在广泛的生物传感器应用。 对于GPCR演示，我们使用标准SPR仪器的工作表明， 去污剂的选择对于获得活性溶解受体是关键的。但标 低通量SPR生物传感器有两个压倒性的缺点：（1）96个样品的分析 溶解条件需要超过两天，并且受体失去显著的 活动在这段时间内，这使得它很难比较所获得的结果， 分析的开始和结束，以及（2）SPR仪器仅限于测试一个 分析缓冲液，这意味着整个测定的成功取决于 分析缓冲液的初始选择。 在第一阶段，我们开发了一个96通道连续流微点（CFM）打印头， 证明了直接从粗制介质将GPCR打印到传感器表面上的能力 使用多达96种不同的分析缓冲液。GPCR也保持湿润和活跃 通过CFM的封闭微通道打印网络在整个打印过程中进行打印。在 在最后的实验中，我们使用192个不同的细胞溶解来自全细胞的GPCR CCR 5。 洗涤剂条件下，并将它们点到SPR传感器表面上，使用我们的CFM 打印头。然后，我们测试了受体的活性，并使用配体结合结果， 确定某种洗涤剂组合最能增强受体活性。运行 使用标准Biacore技术（例如T100）进行分析需要4天 仪器时间相比之下，我们能够在不到2小时内完成分析。 小时 在第二阶段，我们建议把第一阶段的96信道管理系统， SPR成像仪能够以高度并行的格式进行自动化相互作用分析。 以下具体目标详细介绍了瓦萨奇的微流体技术的组合 与商业IBIS SPR成像仪，以产生高通量无标记生物传感器。 1.改进第一阶段的96通道CFM打印头设计， 当安装在IBIS SPR成像仪上时， 2.将96通道CFM安装到IBIS SPR成像仪上，并优化流体特性。 影响平台灵敏度和均匀性的参数。 3.在一台无缝仪器中实现CFM和SPR组件的自动化：自动化 流动池定位、密封、流体处理、阀门、在线脱气和 温度控制CFM和SPR成像仪控制软件的集成， 数据收集/分析软件。 4.演示使用GPCR和抗体的自动化系统。

Project Summary The goal of this Phase II STTR project is to develop a real-time label-free biosensor that can analyze 96 samples at a time, compared to the 6 samples possible with current technologies. This platform will initially be demonstrated with G-protein-coupled receptors (GPCRs) and antibodies. What both applications have in common is the need for higher- throughput sensing, and demonstrating the integrated system for these will illustrate its versatility and potential contributions across the wide spectrum of biosensor applications. For the GPCR demonstration, our work with standard SPR instruments has shown that the choice of detergent(s) is critical for obtaining active solubilized receptor. However, standard low throughput SPR biosensors have two overwhelming drawbacks: (1) The analysis of 96 solubilization conditions requires more than two days and the receptor loses significant activity during this time, which makes it difficult to compare the results obtained at the beginning and end of the analysis and (2) the SPR instrument is limited to testing only one analysis buffer at a time, which means that the success of the entire assay depends on the initial choice of analysis buffer. In Phase I, we developed a 96-channel Continuous Flow Microspotter (CFM) printhead and demonstrated the ability to print GPCRs onto a sensor surface directly from crude media using up to 96 different analysis buffers. The GPCRs were also kept wetted and active throughout the printing process by the CFM's enclosed microchannel printing network. In the final experiment, we solubilized the GPCR CCR5 from whole cells using 192 different detergent conditions and spotted them onto an SPR sensor surface using our CFM printhead. We then tested the activity of the receptor and used the ligand binding results to determine that a certain combination of detergents best enhanced receptor activity. To run this analysis with a standard Biacore technology (e.g. T100) would have required four days of instrument time. In comparison, we were able to perform the analysis in less than 2 hours. In Phase II, we propose to integrate the 96-channel CFM from Phase I with a commercial SPR imager to enable automated interaction analysis in a highly parallel format. The following specific aims detail the combination of Wasatch's microfluidic technologies with the commercial IBIS SPR imager to produce a high-throughput label-free biosensor. 1. Refine the 96-channel CFM printhead design from Phase I to enable optimal performance when mounted on the IBIS SPR imager. 2. Mount the 96-channel CFM onto the IBIS SPR imager and optimize the fluidic parameters that affect platform sensitivity and uniformity. 3. Automate the CFM & SPR components within one seamless instrument: Automation of the flow cell positioning, sealing, fluid handling, valving, in-line degassing and temperature control. Integration of the CFM and SPR imager control software and data collection/analysis software. 4. Demonstrate use of the automated system with GPCRs and antibodies.