Chelated Lanthanide Emission Fingerprinting for Multiplexed Homogeneous NAT

描述（由申请人提供）：多重均相生物测定为生物样品的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，并可大大减少测定周转时间。为了以均质测定形式同时检测多个靶标，靶标特异性荧光标记（在各种配置中，诸如直接标记和信标）是有效的工具。用于多重均相生物测定的其他方法，如本文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，如由于仪器的尺寸或鲁棒性要求而无法使用滤光轮或多个检测器的应用。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光轮而实现的潜在尺寸和耐久性改进来改进多路复用护理点诊断装置。CLEF测定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核酸的灵敏的多重检测。分析完全在溶液中进行，加快了周转时间。CLEF测定使用基于发光寿命的多路复用;不同的靶特异性信标各自具有不同的发光衰减曲线，由与信标缀合的螯合镧系元素确定。所有的信标都用单个LED激发，并且与其互补目标杂交的所有信标发光，而未杂交的那些信标的能量被暗猝灭剂猝灭。所有的发射都是用一个发射过滤器和一个检测器读取的。CLEF方法通过具有新颖的多路复用算法和高速光学器件和数据采集硬件的仪器来实现。CLEF算法将发光衰减分离成由每个信标贡献的分量，确定哪些信标是发信号的，哪些是猝灭的。在这个为期6个月的R43第一阶段，工作是围绕两个具体的目标，旨在证明这种新型的同质多路复用方法的可行性-目标1建立报告化学品和共轭探针，和目标2增强高速仪器和扩展信号处理算法从双工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