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首页> 外文会议>Conference on Molecular Probes for Biomedical Applications; 20080121-22; San Jose,CA(US) >Click chemistry for labeling and detection of biomolecules
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Click chemistry for labeling and detection of biomolecules

机译：单击化学以标记和检测生物分子

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摘要

Described herein is an application of the copper(I)-catalyzed Huisgen [3+2] cycloaddition between azides and alkynes, or click chemistry, for the universal two-step detection of biological macromolecules. The first step involves the metabolic incorporation of an azide or alkyne probe into a macromolecule of interest. The second step involves the click chemistry conjugation of the labeled macromolecule with a partner alkyne or azide-reactive fluorescent probe to form a stable triazole ring conjugate. The fluorescently tagged molecules can be subsequently detected by a number of different fluorescent readout platforms including flow cytometry, fluorescence imaging, and 1-D/2-D gel imaging. We demonstrate application of this technology in two different labeling schemes. First, the labeling of newly synthesized DNA in a novel cell proliferation assay, and second, in the labeling of specific glycoprotein subclasses for biomarker discovery applications. In each case, azide or alkyne probes are introduced metabolically with subsequent detection using click chemistry. Utilization of the cellular enzymatic machinery for high-fidelity target molecule labeling combined with the superior efficiency of click chemistry detection results in highly versatile macromolecular labeling platforms that are unmatched in sensitivity and selectivity.
机译：本文描述了在叠氮化物和炔烃之间进行铜（I）催化的惠斯根[3 + 2]环加成反应或点击化学的应用，该步骤可用于生物大分子的通用两步检测。第一步涉及将叠氮化物或炔烃探针代谢并入目标大分子中。第二步涉及标记的大分子与伴侣炔烃或叠氮化物反应性荧光探针的点击化学缀合，以形成稳定的三唑环缀合物。随后可以通过许多不同的荧光读数平台（包括流式细胞仪，荧光成像和1-D / 2-D凝胶成像）检测荧光标记的分子。我们在两种不同的标记方案中演示了该技术的应用。首先，在新颖的细胞增殖测定中标记新合成的DNA，其次，在用于生物标记物发现应用的特定糖蛋白亚类的标记中。在每种情况下，都会通过点击化学方法将叠氮化物或炔烃探针代谢引入，随后进行检测。利用细胞酶机制进行高保真目标分子标记，再加上点击化学检测的卓越效率，可形成高度通用的大分子标记平台，其灵敏度和选择性无与伦比。

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