Fluorescence-quenching-based enzyme-activity assay by using photon upconversion
Fluorescence-quenching-based enzyme-activity assay by using photon upconversion
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DOI：
10.1002/anie.200705861
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Soukka, Tero
中科院分区：
文献类型：
--
作者：
Rantanen, Terhi;Jaervenpaeae, Marja-Leena;Soukka, Tero
酶活性测定例如用于筛选酶抑制剂和激活剂以发现新的候选药物。[1]基于双标记荧光底物的水解酶的均相测定原理是常用的，并且适用于高通量筛选。这种无分离测定概念依赖于荧光共振能量转移（FRET）的强距离依赖性，其仅在低于10 nm的距离处发生。[2，3]酶的合成内部淬灭底物在分子的一端标记有荧光团，在另一端标记有淬灭剂。当酶与底物反应时，两种标记物被分离，荧光被恢复，但由于生物材料本身的荧光，荧光猝灭法的性能受到限制。这个问题可以通过基于上转换磷光体（UCP）的新型标记技术来解决，[4]其具有在近红外（NIR）激发下在可见波长处光致发光发射的独特性质。在较短的波长下没有检测到自发荧光，因为上转换现象需要自然界中未观察到的连续多光子吸收。由于近红外激发，UCP技术也适用于强烈着色的样品（例如全血），[5]这些样品在紫外和可见波长处吸收，这一过程会干扰其他荧光技术。
Enzyme-activity assays are used, for example, for screening enzyme inhibitors and activators to discover novel drug candidates.[1] A homogeneous assay principle for hydrolyzing enzymes based on a double-labeled fluorogenic substrate is commonly employed and is suitable for high-throughput screening. This separation-free assay concept relies on the strong distance dependency of fluorescence resonance energy transfer (FRET), which takes place only at distances below 10 nm.[2, 3] A synthetic internally quenched substrate for the enzyme is labeled with a fluorophore at one end and a quencher at the other end of the molecule. When the enzyme digests the substrate, the two labels are separated and fluorescence is recovered.The performance of fluorescence-quenching-based homogeneous assays is still limited due to the autofluorescence originating from biological materials. This problem can be solved by a novel label technology based on upconverting phosphors (UCPs),[4] which have the unique property of photoluminescence emission at visible wavelengths under near-infrared (NIR) excitation. No autofluorescence is detected at shorter wavelengths, because the upconversion phenomenon requires sequential multiphoton absorption not observed in nature. Due to the NIR excitation, UCP technology is also applicable to strongly colored samples (for example, whole blood),[5] which absorb at ultraviolet and visible wavelengths, a process that interferes with other fluorescence technologies.