Chelated Lanthanide Emission Fingerprinting for Multiplexed Homogeneous NAT

用于多重均质 NAT 的螯合镧系元素发射指纹图谱

基本信息

批准号：
8454682
负责人：
Amy Droitcour
金额：
$ 16.11万
依托单位：
WAVE 80 BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2014-02-28
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Multiplexed homogeneous bioassays provide an important alternative to microarrays for multiplexed analysis of biological samples, because they avoid some of assay development challenges and can greatly reduce assay turnaround time. For simultaneously detecting multiple targets in a homogeneous assay format, target-specific fluorescent labels (in various configurations, such as directly labeling and beacons) are an effective tool. Additional methods for multiplexed homogeneous bioassays, such as the Chelated Lanthanide Emission Fingerprinting (CLEF) method proposed here, would complement fluorescence methods and potentially offer advantages for some applications, such as those where filter wheels or multiple detectors are impractical due to the size or robustness requirements of the instrument. Multiplexed point of care diagnostic devices are improved by both the rapid turnaround time of homogeneous methods and the potential size and durability improvements achieved by avoiding filter wheels in the proposed multiplexing method. The CLEF assay scheme utilizes the long luminescent lifetimes of chelated lanthanides with molecular beacon technology to provide sensitive, multiplexed detection of nucleic acids. The assay takes place entirely in solution, speeding turnaround time. The CLEF assay uses luminescence-lifetime-based multiplexing; the different target-specific beacons each have different luminescent decay profiles, determined by the chelated lanthanide conjugated to the beacon. All the beacons are excited with a single LED, and all the beacons that are hybridized to their complementary target luminesce, while the energy of those that are not hybridized is quenched by a dark quencher. All emissions are read with a single emission filter and a single detector. The CLEF method is enabled by an instrument with a novel multiplexing algorithm and high-speed optics and data acquisition hardware. The CLEF algorithm separates the luminescent decay into the components contributed by each beacon, determines which beacons are signaling and which are quenched. In this 6-month R43 Phase I, work is organized around two specific aims designed to demonstrate the feasibility of this novel homogeneous multiplexing approach - Aim 1 establishing the reporter chemicals and conjugating them to probes, and Aim 2 enhancing the high-speed instrument and extending signal processing algorithms from duplex to greater degrees of multiplexing. 1

描述（由申请人提供）：多重同质生物测定为生物样品多重分析提供了微阵列的重要替代方案，因为它们避免了一些测定开发挑战，并且可以大大减少测定周转时间。为了以同质测定形式同时检测多个靶标，靶标特异性荧光标记（各种配置，例如直接标记和信标）是一种有效的工具。用于多重均质生物测定的其他方法，例如此处提出的螯合镧系元素发射指纹 (CLEF) 方法，将补充荧光方法，并可能为某些应用提供优势，例如由于尺寸或鲁棒性而导致滤光轮或多个检测器不切实际的应用仪器的要求。通过均质方法的快速周转时间以及通过在所提出的多重方法中避免滤光轮而实现的潜在尺寸和耐用性改进，多重的护理点诊断设备得到了改进。 CLEF 检测方案利用螯合镧系元素的长发光寿命和分子信标技术来提供灵敏的多重核酸检测。该测定完全在溶液中进行，加快了周转时间。 CLEF 测定使用基于发光寿命的多重分析；不同的特定目标信标各自具有不同的发光衰减曲线，这是由与信标共轭的螯合镧系元素决定的。所有信标均由单个 LED 激发，所有与其互补目标发光杂交的信标都会发光，而未杂交的信标的能量则由暗猝灭剂猝灭。所有发射均通过单个发射过滤器和单个检测器读取。 CLEF 方法是通过具有新颖的多重算法以及高速光学和数据采集硬件的仪器来实现的。 CLEF 算法将发光衰减分离为每个信标贡献的分量，确定哪些信标发出信号，哪些信标被淬灭。在为期 6 个月的 R43 第一阶段中，工作围绕两个具体目标进行组织，旨在证明这种新颖的均相多重方法的可行性 - 目标 1 建立报告化学物质并将其与探针结合，目标 2 增强高速仪器和将信号处理算法从双工扩展到更高程度的多路复用。 1