Ratiometric SPCE Detection of miRNA

说明(申请人提供)：荧光技术是生命科学、生物技术、医学诊断、法医学等领域的关键研究工具。基于荧光的测量包括酶联免疫吸附试验、聚合酶链式反应、微阵列基因表达芯片、其他医学诊断、取证测试和生物危害检测技术。在这些技术中的许多技术中通常希望或需要更高的灵敏度，以允许在较小样本体积内可靠地检测较少数量的样本分子。实现这一目标的理想技术不应该需要昂贵的复杂设备。为了检测SPCE荧光，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，采用一种新的比率测量、共聚焦格式来检测Micro-RNA(mi-RNA)类似物。被检测的miRNA是小的，21-23-MERS，参与调节mRNA的功能，引起了许多研究人员的兴趣。SPCE的检测量非常有限，表面受限(约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的高定向荧光、低本底和纯表面激发，检测限(LOD)将比现有方法降低至少10倍。该探测器将是廉价的，并适用于其他系统。总体目标是证明一种新的通用SPCE技术的可行性，该技术使用一种创新的比率测量共聚焦信号收集设备来检测miRNA。这项技术/设备将有助于低成本、高灵敏度地检测复杂生物基质中的mi-RNA，如血浆和细胞提取物。比率测量信号将介于感兴趣样品的荧光团和内部标准荧光团之间。这种比率检测将抵消检测器和样品中的许多变量。这将允许使用计算简单的廉价探测器。其技术目标是：(1)证明波长分辨SPCE是一种敏感和可靠的比率传感技术，具有新的共焦形式(例如针孔)。我们将确定表面结合的寡核苷酸链(作为mi-RNA的类似物)在干净的缓冲液和‘脏基质’(如重组血浆和细胞提取物)中的检测下限。我们预计LOD将分别比溶液和当前最先进的表面技术提高100倍和10倍。(2)开发两种小型、简单、廉价的比率SPCE传感装置的原型(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USB4000探测器的针孔室。在第二阶段，我们将进一步开发待售的原型设备以及能够检测果蝇和其他感兴趣生物表达的miRNAs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。一种名为表面等离子体耦合发射(SPCE)的新表面荧光技术将被开发出来，以一种灵敏、可靠和相对便宜的比率方法来检测寡核苷酸。该装置和方法将在生命科学、医学诊断、法医学等领域中得到应用。

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.