Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging.
Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging.
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DOI：
10.1126/sciadv.1600265
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发表时间：
2016-06
期刊：
影响因子：
13.6
通讯作者：
Miller LW
中科院分区：
文献类型：
--
作者：
Afsari HS;Cardoso Dos Santos M;Lindén S;Chen T;Qiu X;van Bergen En Henegouwen PM;Jennings TL;Susumu K;Medintz IL;Hildebrandt N;Miller LW
时间门控FRET技术用于快速、灵敏的细胞内和细胞外成像。时间门控Förster共振能量转移(FRET)技术采用了长寿命铽配合物(Tb)和半导体量子点(QD)的独特材料组合，为高灵敏和多路生物传感提供了许多优点。虽然时间门控检测可以有效地抑制样品的自发荧光和直接激发的FRET受体的背景荧光，但Tb-to-QD FRET很少被用于生物分子成像。我们展示了可应用于细胞内和细胞外成像的TB到QD时间门控FRET纳米组件。表皮生长因子受体(EGFR)的不同表位与TB和QD结合的抗体和纳米体的免疫染色允许在A431细胞膜上进行有效的TB到QD FRET。TB-to-QD FRET和TB-to-QD-FRET在细胞内的显微注射以及细胞穿透肽介导的HeLa细胞内吞作用进一步证明了其广泛的实用性。FRET从几个TB到同一个QD的有效亮度增强，低纳摩尔浓度的使用，以及快速和灵敏的检测而不需要FRET受体背景荧光，是先进的细胞内外生物分子相互作用成像的重要优势。
The time-gated FRET technique is used for rapid, sensitive intra- and extracellular imaging. Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide–mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.