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首页> 外文期刊>Combinatorial Chemistry & High Throughput Screening >(Research Papers) Time-Resolved Fluorescence Measurements Using Microlens Array and Area Imaging Devices
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(Research Papers) Time-Resolved Fluorescence Measurements Using Microlens Array and Area Imaging Devices

机译：（研究论文）使用微透镜阵列和面积成像设备的时间分辨荧光测量

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Time-resolved fluorescence (TRF) assay formats are frequently used technologies in highthroughput screening. In this article, we have characterised the novel Plate::Vision2 96-microlens array reader (Carl Zeiss Jena GmbH, Germany) and compared it to the novel LEADseeker Generation IV multimodality imaging system (LEADseeker Gen IV;; Amersham Biosciences UK Ltd., UK) for applications in the TRF mode. In europium measurements using the TRF mode, the Plate::Vision displayed a limit of detection for europium of approximately 3 pM, which was comparable to two established TRF readers, the Discovery and the Victor V (both PerkinElmer Life Sciences Inc., USA). The LEADseeker's limit of detection only extended down to europium concentrations of approximately 10 pM in these experiments.nnFor TRF resonance energy transfer (TR-FRET) experiments, a europium-biotin (Eu-biotin) conjugate was titrated with a streptavidin-allophycocyanin (SA-APC) conjugate. The Plate::Vision produced Z' values larger than 0.5 for the acceptor fluorophor emission with concentrations of Eu-biotin as low as 3 nM combined with 175 pM SA-APC. To achieve Z' values of at least 0.5 with the LEADseeker, concentrations of 10 nM Eu-biotin combined with SA-APC of at least 0.8 nM were required. In a drug screening application using TR-FRET, the energy transfer from a europium-labelled protein X (Eu-protein X) to a complex of biotinylated peptide Y with SA-APC was measured. Using the Plate::Vision, a Z' factor larger than 0.5 for the acceptor fluorophor emission was only obtained for a Eu-protein X concentration of at least 10 nM in combination with biotinylated peptide Y / /SA-APC at saturating concentrations.nnBoth the Plate::Vision and the LEADseeker show good quality results for applications in the TRF mode and enable an increased throughput based on their shortened measurement time in comparison to classic photomultiplier tube-based readers.
机译：时间分辨荧光（TRF）分析格式是高通量筛选中常用的技术。在本文中，我们对新型Plate :: Vision2 96微透镜阵列读取器（德国卡尔·蔡司·耶纳有限公司）进行了特性分析，并将其与新型LEADseeker第四代多模态成像系统（LEADseeker Gen IV ；； Amersham Biosciences UK Ltd.，英国）（适用于TRF模式）。在使用TRF模式进行的measurements测量中，Plate :: Vision显示出的a的检出限约为3 pM，可与两个成熟的TRF读卡器Discovery和Victor V（美国PerkinElmer Life Sciences Inc.）媲美。 。在这些实验中，LEADseeker的检出限仅延伸至大约10 pM的。浓度.nn对于TRF共振能量转移（TR-FRET）实验，a-生物素（Eu-生物素）共轭物用链霉亲和素-藻蓝蛋白（SA）滴定-APC）共轭物。对于Eu-生物素浓度低至3 nM和175 pM SA-APC，Plate :: Vision产生的受体荧光发射的Z'值大于0.5。为了使用LEADseeker获得至少0.5的Z'值，需要10 nM的Eu-生物素与SA-APC结合的浓度至少为0.8 nM。在使用TR-FRET的药物筛选应用中，测量了从from标记的蛋白质X（Eu-蛋白质X）到具有SA-APC的生物素化肽Y的复合物的能量转移。使用Plate :: Vision，只有在饱和浓度的生物素化肽Y // SA-APC与至少10 nM的Eu-蛋白质X结合使用时，才能获得大于0.5的受体荧光团发射的Z'因子。 Plate :: Vision和LEADseeker与传统的基于光电倍增管的读取器相比，在TRF模式下的应用显示了良好的质量结果，并且由于缩短了测量时间，因而提高了通量。

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