Cross-talk-free dual-color fluorescence cross-correlation spectroscopy for the study of enzyme activity.
Cross-talk-free dual-color fluorescence cross-correlation spectroscopy for the study of enzyme activity.
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DOI：
10.1021/ac9024768
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发表时间：
2010-02-15
影响因子：
7.4
通讯作者：
Soper, Steven A.
中科院分区：
文献类型：
--
作者：
Lee, Wonbae;Lee, Yong-Ill;Lee, Jeonghoon;Davis, Lloyd M.;Deininger, Prescott;Soper, Steven A.
我们开发了一种无光谱串扰的双色荧光互相关光谱（FCCS）仪器，它为高通量筛选等应用领域的酶活性研究提供了一种读出模式。使用两种不同的激发源同时激发两种光谱上不同的（约250 nm）荧光团Cy 3和IRD 800;一种在532 nm处，另一种在780 nm处。荧光信息在两个不同的颜色通道上处理，用单光子雪崩二极管（SPAD）监测，可以在单分子水平上监测事件。该系统不提供颜色串扰（交叉激发和/或交叉发射）和/或荧光共振能量转移（FRET），显著提高了数据质量。为了提供无串扰操作的证据，使用明亮的微球（λabs = 532 nm，λem= 560 nm）和量子点（λabs = 532 nm，λem= 810 nm）来评估系统。实验结果表明，没有观察到颜色从微球或量子点泄漏到不适当的颜色通道中。为了证明该系统的实用性，使用Cy 3和IRD 800双标记的双链DNA底物，通过FCCS监测APE 1的酶活性，APE 1负责在脱嘌呤/脱嘧啶位点的5′侧切割DNA中的磷酸二酯骨架。在酶的抑制剂（7-硝基吲哚-2-羧酸）存在下，还使用该无串扰FCCS平台监测APE 1的活性。在所有情况下，没有观察到从单分子事件到不适当的颜色通道的光谱泄漏。
We have developed an instrument for spectral cross-talk free dual-color fluorescence cross-correlation spectroscopy (FCCS), which provides a readout modality for the study of enzyme activity in application areas such as high throughput screening. Two spectrally distinct (∼250 nm) fluorophores, Cy3 and IRD800, were excited simultaneously using two different excitation sources; one poised at 532 nm and the other at 780 nm. The fluorescence information was processed on two different color channels monitored with single photon avalanche diodes (SPADs) that could transduce events at the single-molecule level. The system provided no color cross-talk (cross-excitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET), significantly improving data quality. To provide evidence of cross-talk free operation, the system was evaluated using bright microspheres (λabs = 532 nm, λem= 560 nm) and quantum dots (λabs = 532 nm, λem= 810 nm). Experimental results indicated that no color leakage from the microspheres or quantum dots into inappropriate color channels was observed. To demonstrate the utility of the system, the enzymatic activity of APE1, which is responsible for nicking the phosphodiester backbone in DNA on the 5′ side of an apurinic/apyrimidinic site, was monitored by FCCS using a double-stranded DNA substrate dual labeled with Cy3 and IRD800. Activity of APE1 was also monitored in the presence of an inhibitor (7-nitroindole-2-carboxylic acid) of the enzyme using this cross-talk free FCCS platform. In all cases, no spectral leakage from single molecule events into inappropriate color channels was observed.
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DOI：
10.1073/pnas.180317197
发表时间：
2000-09-12
影响因子：
11.1
作者：
Heinze, KG;Koltermann, A;Schwille, P
通讯作者：
Schwille, P
影响因子：
2.8
作者：
Davis, Lloyd M.;Williams, Peter E.;Matayoshi, Edmund D.
通讯作者：
Matayoshi, Edmund D.
分离DNA碱基切除修复的小分子抑制剂。
影响因子：
14.9
作者：
Madhusudan, S;Smart, F;Shrimpton, P;Parsons, JL;Gardiner, L;Houlbrook, S;Talbot, DC;Hammonds, T;Freemont, PA;Sternberg, MJE;Dianov, GL;Hickson, ID
通讯作者：
Hickson, ID
DOI：
10.1073/pnas.2436461100
发表时间：
2004-01-06
影响因子：
11.1
作者：
Kim, SA;Heinze, KG;Schwille, P
通讯作者：
Schwille, P
影响因子：
46.9
作者：
Kogure, T;Karasawa, S;Miyawaki, A
通讯作者：
Miyawaki, A