Use of silver nanostructures on microtransponders in ultra-sensitive assay

描述(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来增强荧光发射，以及(2)提高分析仪的荧光检测灵敏度，从而提高基于微应答器的多路生物检测的灵敏度。该检测的关键元件是MTP，这是一种单片500W 500&lt；m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)、发射逻辑电路和集成天线组成，当发光时，该天线通过变化的磁场发射ID。在分析中，芯片用分子探针衍生，该分子探针能够结合到可以荧光标记的感兴趣的分析物上。芯片在高速、基于流体的分析仪中被读取。分析仪解码MTP ID并测量表面的荧光强度，以索引和报告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金属(通常是银)中的等离子激元形成并通过荧光传递这种能量的现象。在项目过程中，我们将在MTP表面包覆不同类型的银纳米结构，定量和表征MTP表面的荧光增强。此外，分析仪将得到改进，以测量非常低的荧光水平。该项目的目标之一是开发一种基于蛋白质的多重检测方法，以检测前列腺癌(PCA)的尿液生物标记物。选定的生物标志物是AMACR(一种外消旋酶)、endoglin、Annexin A3和细胞因子IL-6。选择生物标记物是因为它们在PCa患者样本中的独特表达谱，而不是在健康对照组中的表达谱。多重MTP检测包括以下步骤：(A)将捕获抗体与银纳米结构包被的微应答器连接，(B)沉积目标抗原标本源，(C)使用相应数量的生物素化抗体和显像剂进行检测。我们将设计和开发检测方法，并针对生物标记物对照标准检测性能。在进行MTP生物标记物分析之前，将使用配置和优化的单个ELISA抗原-抗体分析来优化分析程序。为了分析来自分析仪的数据，我们将修改现有的硬件和软件。 与公共卫生相关：拟议的超灵敏检测系统提供了一种同时进行几项测试的新工具。该系统适用于癌症诊断和其他医疗疾病，提供了一种功能更强大、使用更简单、成本更低的方法。将开发一种诊断前列腺癌的方法，即在疾病的早期阶段以非侵入性的方式检测尿液。该检测还有可能改善PCa疾病的管理。研究人员和临床医生接受基于纳米技术的微应答器上的多重分析，可能会在疾病诊断和治疗、药物发现以及生物或环境研究方面开创新的方法。

DESCRIPTION (provided by applicant): The main goal of the project is to increase the sensitivity of microtransponder-based multiplexed bioassays by (1) introducing a coating with silver nanostructures onto the microtransponders (MTPs) to enhance fluorescence emission, and (2) improving the sensitivity of fluorescence detection of the analyzer. The key element of the assay is the MTP, a monolithic 500 W 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 illuminated, transmit 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 high speed, fluidic\-based analyzer. The analyzer decodes the MTP ID and measures the fluorescence intensity on the surface to index and report information about molecular interactions in the assay. Increased 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 fluorescence enhancement on the surface of the MTP. In addition, the analyzer will be improved to measure very low fluorescence levels. One of the goals of the project is to develop a protein-based, multiplex assay to detect urine biomarkers for prostate cancer (PCa). The selected biomarkers are AMACR (a racemase), endoglin, annexin A3 and cytokine IL-6. The biomarkers were chosen because of their unique expression profile in PCa patient samples versus those in healthy controls. The multiplex MTP assay involves the following steps: (a) conjugation of capture antibodies to silver nanostructure coated microtransponders, (b) deposition of the targeted antigen specimen source, (c) detection using the corresponding number of biotinylated antibodies and a reagent for visualization. We will design and develop the assay, as well as standardize the assay performance against the biomarker controls. Assay procedures will be optimized using individual ELISA antigen-antibody assays configured & optimized before an MTP biomarker assay is performed. In order to analyze the data from the analyzer, we will modify existing hardware and software. PUBLIC HEALTH RELEVANCE: The proposed system for an ultra-sensitive assay provides a new tool for performing several tests at once. The system is applicable to cancer diagnostics and other medical ailments providing a more powerful, simpler to use and less costly approach. An assay will be developed to diagnose prostate cancer by testing urine in an non-invasive way at early phases of the disease. The assay has a potential to also improve PCa disease management. Acceptance of nanotechnology-based multiplex assays on microtransponders by researchers and clinicians could initiate new methods in disease diagnosis and treatment, drug discovery, and biological or environmental research.