Chelated Lanthanide Emission Fingerprinting for Multiplexed Homogeneous NAT

描述(申请人提供)：多路均相生物分析为生物样品的多路分析提供了一种重要的替代微阵列，因为它们避免了一些分析开发挑战，并可以极大地减少分析周转时间。为了以均一的分析格式同时检测多个目标，目标特定的荧光标记(以各种配置，例如直接标记和信标)是一种有效的工具。多路均相生物分析的其他方法，如本文提出的螯合稀土发射指纹(CEF)方法，将补充荧光方法，并有可能为某些应用提供优势，例如，由于仪器的大小或稳定性要求，滤轮或多个检测器是不可行的。通过同质方法的快速周转时间以及通过在所提出的多路复用方法中避免滤轮而实现的潜在尺寸和耐用性改进，多路复用点诊断设备得到了改进。CLEF分析方案利用具有分子信标技术的螯合稀土元素的长发光寿命来提供对核酸的灵敏、多路检测。该分析完全在溶液中进行，加快了周转时间。CLEF分析使用基于发光寿命的多路复用；不同的目标特定信标每个具有不同的发光衰减曲线，由与信标连接的螯合稀土确定。所有的信标都是用一个LED来激发的，所有与其互补的目标发光混合的信标都会发光，而那些没有杂化的信标的能量则会被黑暗猝灭器熄灭。所有的排放物都是用一个发射滤光器和一个探测器读取的。CLEF方法是通过采用新型多路复用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，确定哪些信标在发送信号，哪些信标被熄灭。在为期6个月的R43第一阶段中，围绕两个特定目标组织工作，旨在展示这种新的同质多路复用方法的可行性-目标1建立报告化学物质并将其连接到探针，以及目标2增强高速仪器并将信号处理算法从双工扩展到更大程度的多路复用。1

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