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)在微转发器（MTPs）上引入具有银纳米结构的涂层来增强荧光，以及(2)提高分析仪的荧光检测限来提高基于微转发器的多路生物测定的灵敏度。该检测的关键元件是MTP，这是一个500 x 500 m的单片集成电路芯片，可以以射频（RF）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，当芯片被照亮时，集成天线通过变化的磁场传输ID。在分析中，芯片用分子探针衍生，该分子探针能够与可荧光标记的感兴趣分析物结合。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，以产生有关分析中分子相互作用的信息。金属增强荧光是一种基于光诱导等离子体在金属（通常是银）中形成并通过荧光传输这种能量的现象。在项目过程中，我们将在MTP表面涂覆不同类型的银纳米结构，定量表征MTP硅表面的荧光增强。此外，分析仪将改进，以测量非常低的荧光水平。这将通过(1)使用高灵敏度的低噪声光电倍增管或光子计数装置以及连接的电子电路来实现，(2)引入旨在精确量化非常浅的荧光峰的软件改进，(3)优化光路和对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。优化后的系统性能将得到全面表征。我们预计，与现有的非增强MTP系统相比，该系统的总体灵敏度将提高三阶，这将允许检测存在于低飞摩尔浓度的生物分子，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检测方法来证明该系统的高灵敏度，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的检测。

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.