Ratiometric SPCE Detection of miRNA

荧光技术是生命科学、生物技术、医学诊断、法医学和其他领域的关键研究工具。基于免疫的测量包括ELISA、PCR、微阵列基因表达芯片、其他医学诊断、法医测试和生物危害检测技术。在许多这些技术中，通常期望或需要更高的灵敏度，以允许在较小的样品体积内可靠地检测较少数量的样品分子。理想的技术不需要昂贵的复杂设备。SPCE荧光检测，拟议的项目将使用一种新兴的表面等离子体耦合发射（SPCE）技术，以一种新的比率，共聚焦格式来检测微小RNA（miRNA）类似物。待检测的miRNA是小的，21-23-mer，其参与调节mRNA功能，并且引起许多研究人员的极大兴趣。SPCE的非常有限的表面限制检测体积（~10-18升）为开发非常低背景、高灵敏度但廉价的检测技术创造了机会。通过使用SPCE的高度定向荧光、低背景和仅表面激发，检测限（LOD）将比当前方法降低至少约10倍。该探测器将是廉价和有用的其他系统。总体目标是证明一种新的通用SPCE技术的可行性，该技术使用创新的比率共聚焦信号收集装置来检测miRNA。该技术/装置将可用于低成本、高灵敏度地检测复杂生物基质如血浆和细胞提取物中的mi-RNA。比率信号将在来自感兴趣样品的荧光团和内标荧光团之间。这种比率检测将消除检测器和样品中的许多变量。这将允许使用具有简单计算的廉价检测器。技术目标是：（1）证明波长分辨SPCE是一种灵敏可靠的比率传感技术，具有新的共焦格式（例如针孔）。我们将确定在清洁缓冲液和“脏基质”（如重组血浆和细胞提取物）中表面结合的寡聚DNA链（作为miRNA的类似物）的检测限。我们预计，在LOD的解决方案和当前最先进的表面技术，分别提高100倍和10倍。(2)开发两种小型、简单、廉价的比率式SPCE传感装置原型（图1b和12）。原型将利用激光指示器激发，荧光聚焦到连接到海洋光学USB 4000探测器的针孔室。在第二阶段，我们将进一步开发原型设备，用于沿着销售，该设备具有能够感测果蝇和其他感兴趣的生物体表达的miRNA的阵列。公共卫生相关性：荧光技术是许多领域研究的关键工具。将开发一种称为表面等离子体耦合发射（SPCE）的新表面荧光技术，以灵敏、可靠且相对便宜的比率法检测寡核苷酸。该装置和方法将在生命科学、医学诊断、法医学和其他领域中的应用中是有用的。

DESCRIPTION (provided by applicant): Fluorescence technologies are key research tools in the life sciences, biotechnology, medical diagnostics, forensics, and other fields. Fluorescence-based measurements include ELISA, PCR, microarray gene expression chips, other medical diagnostics, forensics tests and biohazard detection technologies. More sensitivity is often desired or needed in many of these techniques to permit reliable detection of a smaller number of sample molecules within in a smaller sample volume. The ideal technology for this should not require expensive complicated equipment. Detection of SPCE Fluorescence, the proposed project will use an emerging surface plasmon- coupled emission (SPCE) technology in a novel ratiometric, confocal format to detect micro-RNA (mi-RNA) analogs. The miRNA to be detected are small, 21-23-mers that are involved in regulating mRNA function and are of considerable interest to many researchers. The very limited, surface confined detection volumes of SPCE (~10-18 liters) create an opportunity to develop a very low background, highly sensitive, yet inexpensive detection technology. The limit of detection (LOD) will be reduced by at least ~10-fold over current methodologies through the use of highly directional fluorescence, low background, and surface only excitation of SPCE. The detector will be inexpensive and useful for other systems. The general objectives are to demonstrate the feasibility a new generic SPCE technology using an innovative ratiometric confocal signal collection device for the detection of miRNA. This technique/device will be useful for low-cost, highly sensitive detection of mi-RNA in complex biological matrixes such as plasma and cell extracts. The ratiometric signal will be between a fluorophore from the sample of interest and an internal standard fluorophore. This ratiometric detection will cancel out many variables in the detector and samples. This will allow for the use of inexpensive detectors with simple calculations. The technical objectives are: (1) To demonstrate that wavelength-resolved SPCE is a sensitive and reliable technology for ratiometric sensing with a novel confocal format (e.g. pinhole). We will determine the detection limits of surface bound oligo-DNA strands (as analogs of mi-RNA) in clean buffers and in a 'dirty matrix' such as reconstituted plasma and cell extracts. We expect improvements of 100-fold and 10-fold in the LOD over solution and current state of the art surface techniques, respectively. (2) To develop two prototypes of a small, simple, inexpensive ratiometric SPCE sensing device (Fig. 1b and 12). Prototypes will utilize laser pointer excitation with fluorescence focused to a pinhole chamber connected to an Ocean Optics USB4000 detector. In Phase II we will further develop the prototype device for sale along with an array capable of sensing miRNAs expressed by the fruit fly and other organisms of interest. PUBLIC HEALTH RELEVANCE: Fluorescence technologies are key tools for research in many areas. A new surface fluorescence technology called Surface Plasmon Coupled Emission (SPCE) will be developed to sense oligonucleotides in a ratiometric method that will be sensitive, reliable and relatively inexpensive. The device and method will be useful in applications in the life sciences, medical diagnostics, forensics and other fields.