Li Yan-Ran, Liu Qian, Hong Zhangyong, Wang He-Fang
College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology (Nankai University), Tianjin Key Laboratory of Molecular Recognition and Biosensing, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University , 94 Weijin Road, Tianjin 30071, China.
Anal Chem. 2015 Dec 15;87(24):12183-9. doi: 10.1021/acs.analchem.5b03064. Epub 2015 Nov 23.
For the widely used "off-on" fluorescence (or phosphorescence) resonance energy transfer (FRET or PRET) system, the separation of donors and acceptors species was vital for enhancing the sensitivity. To date, separation of free donors from FRET/PRET inhibition systems was somewhat not convenient, whereas separation of the target-induced far-between acceptors has hardly been reported yet. We presented here a novel magnetic separation-assistant fluorescence resonance energy transfer (MS-FRET) inhibition strategy for highly sensitive detection of nucleolin using Cy5.5-AS1411 as the donor and Fe3O4-polypyrrole core-shell (Fe3O4@PPY) nanoparticles as the NIR quenching acceptor. Due to hydrophobic interaction and π-π stacking of AS1411 and PPY, Cy5.5-AS1411 was bound onto the surface of Fe3O4@PPY, resulting in 90% of fluorescence quenching of Cy5.5-AS1411. Owing to the much stronger specific interaction of AS1411 and nucleolin, the presence of nucleolin could take Cy5.5-AS1411 apart from Fe3O4@PPY and restore the fluorescence of Cy5.5-AS1411. The superparamagnetism of Fe3O4@PPY enabled all separations and fluorescence measurements complete in the same quartz cell, and thus allowed the convenient but accurate comparison of the sensitivity and fluorescence recovery in the cases of separation or nonseparation. Compared to nonseparation FRET inhibition, the separation of free Cy5.5-AS1411 from Cy5.5-AS1411-Fe3O4@PPY solution (the first magnetic separation, MS-1) had as high as 25-fold enhancement of the sensitivity, whereas further separation of the nucleolin-inducing far-between Fe3O4@PPY from the FRET inhibition solution (the second magnetic separation, MS-2) could further enhance the sensitivity to 35-fold. Finally, the MS-FRET inhibition assay displayed the linear range of 0.625-27.5 μg L(-1) (8.1-359 pM) and detection limit of 0.04 μg L(-1) (0.05 pM) of nucleolin. The fluorescence intensity recovery (the percentage ratio of the final restoring fluorescence intensity to the quenched fluorescence intensity of Cy5.5-AS1411 solution by 0.09 g L(-1) Fe3O4@PPY) was enhanced from 36% (for nonseparation) to 56% (for two magnetic separations). This is the first accurate evaluation for the effect of separating donor/acceptor species on the FRET inhibition assay.
对于广泛应用的“开启-关闭”荧光（或磷光）共振能量转移（FRET或PRET）系统而言，供体和受体物种的分离对于提高灵敏度至关重要。迄今为止，从FRET/PRET抑制系统中分离游离供体有些不便，而目标诱导的远距离受体的分离尚未见报道。在此，我们提出了一种新型的磁分离辅助荧光共振能量转移（MS-FRET）抑制策略，用于以Cy5.5-AS1411作为供体、Fe3O4-聚吡咯核壳（Fe3O4@PPY）纳米颗粒作为近红外猝灭受体来高灵敏度检测核仁素。由于AS1411与PPY之间的疏水相互作用和π-π堆积，Cy5.5-AS1411结合到Fe3O4@PPY表面，导致Cy5.5-AS1411的荧光猝灭90%。由于AS1411与核仁素之间具有更强的特异性相互作用，核仁素的存在可使Cy5.5-AS1411与Fe3O4@PPY分离，并恢复Cy5.5-AS1411的荧光。Fe3O4@PPY的超顺磁性使得所有分离和荧光测量都能在同一个石英池中完成，从而能够方便而准确地比较分离或未分离情况下的灵敏度和荧光恢复情况。与非分离FRET抑制相比，从Cy5.5-AS1411-Fe3O4@PPY溶液中分离游离的Cy5.5-AS1411（第一次磁分离，MS-1）可使灵敏度提高高达25倍，而从FRET抑制溶液中进一步分离核仁素诱导的远距离Fe3O4@PPY（第二次磁分离，MS-2）可将灵敏度进一步提高到35倍。最终，MS-FRET抑制分析显示核仁素的线性范围为0.625 - 27.5 μg L(-1)（8.1 - 359 pM），检测限为0.04 μg L(-1)（0.05 pM）。荧光强度恢复率（最终恢复的荧光强度与0.09 g L(-1) Fe3O4@PPY使Cy5.5-AS1411溶液猝灭的荧光强度的百分比）从36%（非分离情况）提高到56%（两次磁分离情况）。这是对供体/受体物种分离对FRET抑制分析影响的首次准确评估。
