Confocal Nanoscanning, Bead Picking (CONA): PickoScreen Microscopes for Automated and Quantitative Screening of One-Bead One-Compound Libraries
Confocal Nanoscanning, Bead Picking (CONA): PickoScreen Microscopes for Automated and Quantitative Screening of One-Bead One-Compound Libraries
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DOI：
10.1021/cc900059q
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发表时间：
2009-09-01
影响因子：
--
通讯作者：
Auer, Manfred
中科院分区：
文献类型：
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作者：
Hintersteiner, Martin;Buehler, Christof;Auer, Manfred
固相组合化学可以快速且经济高效地访问基于微珠的大型库，其化合物数量轻松超过数万种化合物。将单珠一种化合物库珠与荧光标记的靶标一起孵育。蛋白质以及识别和分离含有结合目标蛋白质的珠子，可能代表最强大的通用初级高通量筛选形式之一。基于这种检测原理的珠上筛选（OBS）可以在有限的自动化下进行。通常，命中珠检测（即识别与珠上的化合物结合的荧光标记蛋白的头）依赖于在宽视野显微镜下的目视检查。使用低分辨率检测技术，命中珠的识别及其排名受到低荧光信号强度和库珠自身荧光水平不同的限制。为了充分发挥所有 OBS 过程的潜力，需要可靠的方法来自动定量检测命中珠及其随后的分离。在与 Evotec Technologies（现为 Perkin-Elmer Cellular Technologies 德国有限公司）的共同合作中，我们构建了两个共焦珠扫描仪和拾取器平台 PS02 以及专用于自动化高分辨率 OBS 的高速变型 PS04。 PSOX 仪器将标准 MTP 板底部珠子单层的全自动共焦大面积扫描与通过液压驱动拾取器毛细管半自动分离单个命中珠子相结合。在每个珠子的赤道平面上以高空间分辨率对荧光强度进行量化，可以可靠地区分完全明亮的自发荧光珠子和真实的命中珠子，后者在珠子外部几微米处表现出增强的荧光信号。在不到 7 小时内实现了高达 200 000 个珠子的筛选速度和类似于 1 个珠子/分钟的拾取时间，使得能够以高灵敏度、准确度和速度开发一珠一化合物文库。
Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target. proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing heads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope, Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of all OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PSOX instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution ill the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200 000 bead assayed in less than 7 h and the picking time of similar to 1 bead/min allow exploitation of one-bead one-compound libraries with high sensitivity, accuracy, and speed.