Time-Resolved FRET Biosensor Based on Amine-Functionalized Lanthanide-Doped NaYF4 Nanocrystals
Time-Resolved FRET Biosensor Based on Amine-Functionalized Lanthanide-Doped NaYF4 Nanocrystals
复制标题
基于胺功能化镧系元素掺杂 NaYF4 纳米晶体的时间分辨 FRET 生物传感器
DOI：
10.1002/anie.201100303
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发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Chen, Xueyuan
中科院分区：
文献类型：
--
作者：
Tu, Datao;Liu, Liqin;Chen, Xueyuan
近年来，掺杂镧系元素（Ln 3+）的纳米晶体（NC）由于其具有尖锐的f-f发射峰、长的光致发光（PL）寿命和大的Stokes位移而成为生物学应用的功能纳米材料的前沿。[1]与生物成像和生物检测中常用的传统有机荧光团和量子点（QD）相比，Ln 3+掺杂的纳米碳显示出上级特性，例如高化学稳定性、高抗光漂白性和低毒性。[2]一个显着的应用Ln3+掺杂的NC是基于荧光共振能量转移（FRET）的新型发光生物传感器的发展，一个非辐射的过程，其特征在于激发供体荧光团（例如，Ln3+掺杂的NC）和受体荧光团（例如，有机染料）之间的能量转移通过长程偶极-偶极相互作用。[3]供体和受体荧光团通过生物缀合紧密连接，通常小于几纳米。[3b]这种能量转移过程可以通过监测供体激发后受体和供体的PL发射光谱来检测。PL发射强度的变化对引入用于供体和受体之间的生物缀合的靶生物分子的浓度敏感。[4]不幸的是，对于具有稳态检测模式的传统FRET测定，自发荧光干扰严重损害了荧光检测的灵敏度，这限制了涉及有机染料、Ln 3+螯合物和量子点等紫外激发生物探针的FRET技术的实际应用。为了避免自发荧光和提高检测灵敏度，出现了一些新的FRET检测技术。例如，上转换FRET（UC-FRET）测定已经被引入作为用于生物样品的定量检测的良好候选，由此UC磷光体在近红外（NIR）区域中被激发以实现可见光发射，使得生物化合物不产生自发荧光。[5]时间分辨FRET（TR-FRET）采用镧系离子如Tb 3+和Eu 3+的长寿命PL是另一种有效的策略，以完全消除来自细胞和组织的散射光和自发荧光的干扰，如先前提出的用于分子探针如Ln 3+螯合物。[6]典型的镧系元素络合物的PL寿命大约为几毫秒或更长，[7]与常见的有机染料和生物化合物形成鲜明对比，它们通常在纳秒范围内。[4]在TR-FRET分析中，来自供体如Ln 3+螯合物的能量转移将明显延长受体如有机染料的PL寿命，其本质上是短寿命的，[6c]由于受体激发态从长寿命Ln 3+激发态的缓慢布居。根据这一原理，通过设置适当的延迟时间和选通时间，可以测量TRFET信号，而不受短寿命背景的干扰（图1）。与常规FRET相比，该方法在发光生物检测中提供了具有显著高信噪比的信号。到目前为止，基于Ln 3+螯合物的分子探针已被开发为各种免疫测定中的TR-FRET生物探针，因为它们的长PL寿命（通常长于1 ms）和大斯托克斯位移（> 200 nm）。然而，大多数Ln 3+螯合物在强烈和连续的激发下容易发生光漂白。[8]在此，我们提出了基于胺官能化的Ln 3+掺杂的NCs的TR-FRET生物传感的第一个演示。与Ln 3+螯合物相反，
Recently, nanocrystals (NCs) doped with lanthanides (Ln3+) have come to the forefront of functional nanomaterials for a variety of biological applications due to their sharp f–f emission peaks, long photoluminescence (PL) lifetimes, and large Stokes shifts.[1] Compared to traditional organic fluorophores and quantum dots (QDs) commonly used in bioimaging and biodetection, Ln3+-doped NCs show superior features such as high chemical stability, high resistance to photobleaching, and low toxicity.[2] A significant application of Ln3+-doped NCs is the development of novel luminescent biosensors based on fluorescence resonance energy transfer (FRET), a nonradiative process characterized by energy transfer between an excited donor fluorophore (eg, Ln3+-doped NC) and an acceptor fluorophore (eg, organic dye) through long-range dipole–dipole interactions.[3] The donor and acceptor fluorophores are linked in close proximity, typically less than a few nanometers, through bioconjugation.[3b] This energy-transfer process can be detected by monitoring the PL emission spectra of acceptor and donor upon excitation of the donor. The change in PL emission intensity is sensitive to the concentration of target biomolecules introduced for bioconjugation between donor and acceptor.[4] Unfortunately, for conventional FRET assays with steady-state detection mode, the sensitivity of fluorescence detection is severely compromised by autofluorescence interference, which limits practical application of the FRET technique involving UV-excited bioprobes such as organic dyes, Ln3+ chelates, and QDs. To avoid autofluorescence and improve detection sensitivity, some novel FRET assay techniques have emerged. For instance, upconversion FRET (UC-FRET) assay has been introduced as a good candidate for quantitative detection of biological samples, whereby UC phosphors are excited in the near-infrared (NIR) region to achieve visible emissions, so that no autofluorescence is produced from biocompounds.[5] Time-resolved FRET (TR-FRET) employing the long-lived PL of lanthanide ions such as Tb3+ and Eu3+ is another effective strategy to completely eliminate the interference of scattered light and autofluorescence from cells and tissues, as previously proposed for molecular probes such as Ln3+ chelates.[6] The PL lifetime of typical lanthanide complexes is on the order of a few milliseconds or longer,[7] in sharp contrast to those of common organic dyes and biocompounds, which lie typically in the nanosecond range.[4] In a TR-FRET analysis, energy transfer from the donors such as Ln3+ chelates will apparently lengthen the PL lifetime of acceptors such as organic dyes, which are intrinsically short-lived,[6c] due to slow population of the acceptor excited state from the longlived Ln3+ excited state. On the basis of this principle, the TRFRET signal can be measured free of the interference of short-lived background by setting appropriate delay time and gate time (Figure1). This method offers a signal with remarkably high signal-to-noise ratio in luminescent biodetection as compared to conventional FRET. Thus far, molecular probes based on Ln3+ chelates have been developed as TR-FRET bioprobes in various immunoassays because of their long PL lifetimes, often longer than 1 ms, and large Stokes shifts (> 200nm). However, most Ln3+ chelates are susceptible to photobleaching under intense and continuous excitation.[8] Herein we present the first demonstration of TR-FRET biosensing based on aminefunctionalized Ln3+-doped NCs. As opposed to Ln3+ chelates,