Multiplexed GPCR Characterization Using SPR

描述（由申请人提供）：这个第二阶段STTR项目的目标是开发一种实时无标签生物传感器，可以一次分析96个样品，而当前技术可能分析6个样品。该平台将首先用g蛋白偶联受体（gpcr）和抗体进行演示。这两种应用的共同点是需要更高通量的传感，并且展示这些集成系统将说明它的多功能性和在广泛的生物传感器应用中的潜在贡献。对于GPCR演示，我们使用标准SPR仪器的工作表明，洗涤剂的选择对于获得活性溶解受体至关重要。然而，标准的低通量SPR生物传感器有两个压倒性的缺点：(1) 96种增溶条件的分析需要两天以上的时间，在此期间受体失去了显著的活性，这使得分析开始和结束时获得的结果难以比较；(2)SPR仪器一次只能测试一种分析缓冲液，这意味着整个分析的成功取决于分析缓冲液的初始选择。在第一阶段，我们开发了一种96通道连续流微点（CFM）打印头，并演示了使用多达96种不同的分析缓冲液直接从粗介质将gpcr打印到传感器表面的能力。通过CFM封闭的微通道打印网络，gpcr在整个打印过程中也保持湿润和活跃。在最后的实验中，我们使用192种不同的洗涤剂条件从整个细胞中溶解GPCR CCR5，并使用CFM打印头将其标记到SPR传感器表面。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的组合可以最好地增强受体的活性。如果使用标准Biacore技术（例如T100）进行分析，则需要4天的仪器时间。相比之下，我们能够在不到2小时的时间内完成分析。在第二阶段，我们建议将第一阶段的96通道CFM与商业SPR成像仪集成在一起，以实现高度并行格式的自动交互分析。以下具体目标详细介绍了Wasatch的微流体技术与商用IBIS SPR成像仪的结合，以生产高通量无标签生物传感器。1. 改进第一阶段的96通道CFM打印头设计，以便在安装在IBIS SPR成像仪上时实现最佳性能。2. 将96通道CFM安装到IBIS SPR成像仪上，优化影响平台灵敏度和均匀性的流体参数。3. 在一个无缝仪器中自动化CFM和SPR组件：自动化流池定位，密封，流体处理，阀门，在线脱气和温度控制。CFM和SPR成像仪控制软件和数据收集/分析软件的集成。4. 演示GPCRs和抗体自动化系统的使用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Antibody analyses are one of the most common applications of biosensor technology and are typically straightforward. GPCRs are the hottest and most challenging system that biosensor users are tackling. Up to half of the drugs on the market today modulate some form of GPCR activity, and it is estimated that 25-50% of the total drug targets are in the GPCR families. GPCRs are the most studied of the major drug target classes, yet they are challenging to work with because they are normally membrane associated, present in low abundance, and unstable. By enabling the high throughput study of GPCRs, there is enormous potential for speeding drug development, treatments, and the associated health of patients with hundreds of different diseases.