Multiplexed GPCR Characterization Using SPR

描述（由申请人提供）：该第二阶段STTR项目的目标是开发一种实时无标记生物传感器，该生物传感器可以一次分析96个样品，而当前技术只能分析6个样品。该平台最初将用G蛋白偶联受体（GPCR）和抗体来证明。这两种应用的共同点是需要更高的通量传感，并展示这些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，去污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生物传感器具有两个压倒性的缺点：（1）96种溶解条件的分析需要两天以上，并且受体在这段时间内失去显著的活性，这使得难以比较在分析开始和结束时获得的结果，和（2）SPR仪器限于一次仅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，我们开发了一个96通道连续流微量点样仪（CFM）打印头，并证明了使用多达96种不同的分析缓冲液直接从原始介质将GPCR打印到传感器表面的能力。通过CFM的封闭微通道印刷网络，GPCR在整个印刷过程中也保持湿润和活性。在最后的实验中，我们使用192种不同的洗涤剂条件溶解来自全细胞的GPCR CCR 5，并使用我们的CFM打印头将它们点样到SPR传感器表面上。然后，我们测试了受体的活性，并使用配体结合结果来确定某种去污剂组合最好地增强了受体活性。使用标准Biacore技术（例如T100）运行此分析将需要四天的仪器时间。相比之下，我们能够在不到2小时内完成分析。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，以实现高度并行格式的自动化相互作用分析。以下具体目标详细说明了Wasatch的微流体技术与商业IBIS SPR成像仪的组合，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，使其在安装在IBIS SPR成像仪上时具有最佳性能。2.将96通道CFM安装到IBIS SPR成像仪上，并优化影响平台灵敏度和均匀性的流体参数。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成像仪控制软件和数据收集/分析软件的集成。4.演示使用GPCR和抗体的自动化系统。 公共卫生相关性：抗体分析是生物传感器技术最常见的应用之一，通常很简单。GPCR是生物传感器用户正在解决的最热门和最具挑战性的系统。目前市场上多达一半的药物调节某种形式的GPCR活性，据估计，总药物靶标的25-50%在GPCR家族中。GPCR是研究最多的主要药物靶标类别，但它们具有挑战性，因为它们通常与膜相关，丰度低，并且不稳定。通过实现GPCR的高通量研究，在加速药物开发、治疗和数百种不同疾病患者的相关健康方面具有巨大的潜力。

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.