Multiplexed GPCR Characterization Using SPR

项目摘要 这一二期STTR项目的目标是开发一种实时无标记生物传感器，它可以 一次分析96个样本，而当前可能分析6个样本 技术。这一平台将首先通过G蛋白偶联受体进行演示 (GPCRs)和抗体。这两个应用程序的共同点是需要更高的 吞吐量感测，并演示针对这些的集成系统将说明其 在生物传感器应用的广泛范围内的多功能性和潜在贡献。 对于GPCR演示，我们使用标准SPR仪器的工作表明 洗涤剂的选择(S)是获得活性增溶受体的关键。然而，标准 低通量SPR生物传感器有两个突出的缺点：(1)对96个 溶解条件需要两天以上，受体显著丧失。 这段时间内的活动，这使得很难比较在 分析的开始和结束以及(2)SPR仪器仅限于测试一个 一次分析缓冲，这意味着整个分析的成功取决于 分析缓冲区的初始选择。 在第一阶段，我们开发了96通道连续流动微观测仪(CFM)打印头和 演示了直接从原始介质将GPCR打印到传感器表面的能力 使用多达96种不同的分析缓冲区。GPCR也保持湿润和活跃。 整个印刷过程由CFM的封闭式微通道印刷网络完成。在……里面 在最后的实验中，我们从整个细胞中溶解了GPCRCCR5，使用了192个不同的 洗涤剂的条件，并使用我们的CFM将它们点到SPR传感器表面 打印头。然后我们测试了受体的活性，并使用配体结合的结果来 确定某种洗涤剂组合最能增强受体活性。奔跑 使用标准Biacore技术(例如T100)进行此分析需要四天时间 乐器时间。相比之下，我们能够在不到2分钟的时间内完成分析 几个小时。 在第二阶段，我们建议将第一阶段的96声道CFM与商业 SPR成像仪以高度并行的格式实现自动交互分析。 以下具体目标详细说明了Wasatch的微流体技术的组合 与商用的IBIS SPR成像仪一起生产高通量的无标记生物传感器。 1.从第一阶段开始改进96通道CFM打印头设计，以实现最佳 安装在IBIS SPR成像器上时的性能。 2.将96通道CFM安装到IBIS SPR成像仪上，并对流体进行了优化 影响平台灵敏度和一致性的参数。 3.在一个无缝仪器中实现CFM和SPR组件的自动化：自动化 流动单元的定位、密封、流体处理、阀门、在线脱气和 温度控制。集成CFM和SPR成像仪控制软件和 数据收集/分析软件。 4.演示使用具有GPCRs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.