SPEI Biosensor Development and Optimization

描述（由申请人提供）：生物传感器是研究和临床诊断的重要工具。由于生物传感器的应用范围很广，要求也各不相同，理想的生物传感器技术将结合灵敏度和易于复用，并且无需标签即可对分析物进行检测和定量。它还将与许多不同的捕获剂（例如抗体、适体、肽）兼容，并为收集动力学和平衡结合数据提供实时检测。目前可用的生物传感器不能满足所有这些苛刻的标准。在这里，我们提出一种基于表面等离子体增强照明（SPEI）的生物传感器解决了这些问题，因此有望在基础生物学、转化研究和临床诊断领域产生广泛的影响。我们在本提案中描述的SPEI生物传感器是基于测量通过金属表面纳米级（50-200nm）孔径的光透射量的变化。我们和其他人已经发现，透射光的量在很大程度上取决于传感器表面的局部折射率，该折射率在分析物与固定捕获试剂结合时发生变化。虽然基于表面等离子体共振（SPR）的生物传感器也可以检测由于生物分子相互作用而引起的局部折射率的变化，但等离子体激发方法对于SPEI技术来说是非常不同的。这些差异，在本提案中详细描述，允许有源传感面积非常小，实现非常高的多路复用程度，卓越的分辨率，并增加灵敏度至少20倍于光栅耦合SPR。然而，与传统SPR的相似性使得为传统SPR完成的大部分应用程序工作可以与SPEI一起使用。我们建议设计和制造一种多路SPEI生物传感器，并通过检测和定量复杂介质中的多种细胞因子来证明其性能。将解决的具体问题包括开发适当的表面化学以减少非特异性结合，开发能够准确和同时定量多种分析物的方法，以及开发优化检测特异性（减少假阳性发生率）的方法。

DESCRIPTION (provided by applicant): Biosensors are important tools for both research and clinical diagnostics. Since the range of applications for biosensors is broad and the requirements varied, the ideal biosensor technology would combine sensitivity with facile multiplexing and enable the detection and quantification of analytes without the need for a label. It would also be compatible with many different capture agents (e.g. antibodies, aptamers, peptides) and provide real-time detection for the collection of kinetic, as well as equilibrium, binding data. Currently available biosensors do not meet all of these demanding criteria. Here, we propose that a biosensor based on surface plasmon enhanced illumination (SPEI) addresses these issues and thus is expected to have a broad impact in the areas of basic biology, translational research, and clinical diagnostics. The SPEI biosensor that we describe in this proposal is based on measuring changes in the amount of light transmitted through nanoscale (50-200nm) apertures in a metallic surface. We and others have found that the amount of transmitted light depends strongly on the local index of refraction at the sensor surface, which is altered upon the binding of analytes to immobilized capture reagents. While biosensors based on surface plasmon resonance (SPR) also detect changes in the local index of refraction due to biomolecular interactions, the plasmon excitation method is very different for-the SPEI technology. These differences, described in detail in this proposal, allow the active sensing area to be very small, enabling a very high degree of multiplexing, superior resolution, and an increased sensitivity of at least 20 fold over grating coupled SPR. The similarity to conventional SPR, however, allows for the body of applications work done for conventional SPR to be used with SPEI. We propose to design and fabricate a multiplexed SPEI biosensor and demonstrate its performance by detecting and quantifying multiple cytokines in complex media. The specific issues that will be addressed include developing appropriate surface chemistry to reduce nonspecific binding, developing methods that enable the accurate and simultaneous quantification of multiple analytes, and developing methods to optimize the specificity of detection (reduce the incidence of false positives).