Use of Silver Nanostructures on Microtransponders in Ultra-sensitive Assay

描述（由申请人提供）：该项目的主要目标是通过（1）在微应答器（MTP）上引入具有银纳米结构的涂层以增强荧光，以及（2）提高分析仪的荧光检测限来提高基于微应答器的多重生物测定的灵敏度。该检测的关键元件是MTP，这是一种单片500 x 500 m集成电路芯片，可以在射频（RF）下传输其识别码。每个芯片由光电池、只读存储器（ROM）、传输逻辑电路和集成天线组成，当芯片被照射时，集成天线通过变化的磁场传输ID。在测定中，芯片用能够结合可被荧光标记的感兴趣的分析物的分子探针衍生化。芯片在基于流体的分析仪中读取。分析仪解码MTP ID并测量MTP表面上的荧光强度，以产生有关测定中分子相互作用的信息。金属增强荧光是基于金属（通常为银）中的光诱导等离子体激元形成以及该能量通过荧光的传输的现象。在项目过程中，我们将用不同类型的银纳米结构涂覆MTP，定量和表征MTP硅表面上的荧光增强。此外，分析仪将得到改进，以测量非常低的荧光水平。这将通过以下方式实现：（1）使用高灵敏度低噪声光电倍增管或光子计数装置和连接的电子电路，（2）对旨在精确定量非常浅的荧光峰的软件进行改进，（3）优化光学路径和光学系统所有组件的关键分析，以及（4）增加激发光束的功率。优化系统的性能将被彻底表征。我们预计整体的三阶灵敏度改善现有的非增强MTP系统，这将允许检测的生物分子存在于低飞摩尔浓度的测定中，其中形成三明治的分子具有足够高的亲和力。还将开发几种类型的测定来证明系统的高灵敏度，包括白细胞介素IL-2和IL-6、mi RNA和癌症标志物细胞色素c和Bax的测定。

DESCRIPTION (provided by applicant): The main goal of the project is to increase sensitivity of the microtransponder-based multiplexed bioassays by (1) introducing a coating with silver nanostructures onto the microtransponders (MTPs) to enhance fluorescence, and (2) improving the fluorescence detection limit of the analyzer. The key element of the assay is the MTP, a monolithic 500 x 500 m integrated circuit chip that can transmit its identification code at a radio frequency (RF). Each chip consists of photocells, read-only memory (ROM), transmit logic circuitry, and an integrated antenna which, when the chip is illuminated, transmits an ID through a varying magnetic field. In an assay, the chips are derivatized with a molecular probe capable of binding to an analyte of interest that can be fluorescently labeled. The chips are read in a fluidics-based analyzer. The analyzer decodes the MTP ID and measures the fluorescence intensity on the MTP surface to yield information about molecular interactions in the assay. Metal-enhanced fluorescence is a phenomenon based on light-induced plasmon formation in the metal, often silver, and transmission of this energy through fluorescence. During the course of the project, we will coat MTPs with different types of sliver nanostructures, quantitate and characterize the fluorescence enhancement on the silicon surface of the MTP. In addition, the analyzer will be improved to measure very low fluorescence levels. This will be achieved by (1) using a highly sensitive low-noise photomultiplier or photon counting device and the connected electronics circuitry, (2) introducing improvements to software aimed at precise quantification of very shallow fluorescence peaks, (3) optimizing the optical path and the critical analysis of all components of the optical system, and (4) increasing the power of the excitation beam. The performance of the optimized system will be thoroughly characterized. We anticipate an overall three-order sensitivity improvement over the existing non-enhanced MTP system, which would allow for detection of biomolecules present in low femtomolar concentrations in assays where the molecules forming a sandwich have sufficiently high affinity. Several types of assays will also be developed to demonstrate the high sensitivity of the system, including assays for interleukins IL-2 and IL-6, mi RNAs and cancer markers cytochrome c and Bax.