Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Forster Resonance Energy Transfer Relays: Characterization and Biosensing
Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Forster Resonance Energy Transfer Relays: Characterization and Biosensing
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DOI：
10.1021/ja210162f
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发表时间：
2012-01-25
影响因子：
15
通讯作者：
Medintz, Igor L.
中科院分区：
文献类型：
--
作者：
Algar, W. Russ;Wegner, David;Medintz, Igor L.
半导体量子点（QD）生物共轭物独特的电子物理性质为主动传感、成像和光学诊断提供了许多优势。特别是，量子点已被广泛采用作为供体或受体的福斯特共振能量转移（FRET）为基础的测定和生物传感器。在这里，我们扩大了他们的效用，证明量子点可以同时作为受体和捐助者的时间门控FRET继电器的作用。为了实现这种配置，QD被用作中心纳米平台，并与用长寿命发光铽（III）络合物（Tb）或荧光染料Alexa Fluor 647（A647）标记的肽或寡核苷酸共组装。在FRET中继中，QD充当关键中间体，其中（1）激发态Tb供体在适当的微秒延迟后将能量转移到基态QD，并且（2）QD随后将该能量转移到A647受体。对FRET中继的每个步骤进行详细的生物物理分析。发现增加Tb/QD比率的组装线性地增加FRET敏化的时间门控QD光致发光强度的大小。重要的是，发现Tb敏化随后的QD A647供体受体FRET对，而不显著影响中继中第二步内的固有能量转移效率。将量子点纳入这种类型的时间门控能量转移配置的效用在用于监测蛋白酶活性和核酸杂交的原型生物测定中得到证明;后者包括双靶格式，其中每个正交FRET步骤转导单独的结合事件。这种时间门控FRET方法的潜在益处包括：消除背景荧光，在单个QD-生物缀合物中访问两个近似独立的FRET机制，以及基于QD-FRET的光谱时间分辨率而不需要QD的多种颜色的多重生物传感。
The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Forster resonance energy transfer (FRET)based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD A647 donor acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.