Paper-Based Solid-Phase Multiplexed Nucleic Acid Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As Donors in Fluorescence Resonance Energy Transfer
Paper-Based Solid-Phase Multiplexed Nucleic Acid Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As Donors in Fluorescence Resonance Energy Transfer
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DOI：
10.1021/ac401471n
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发表时间：
2013-08-06
影响因子：
7.4
通讯作者：
Krull, Ulrich J.
中科院分区：
文献类型：
--
作者：
Noor, M. Omair;Krull, Ulrich J.
本文提出了一种基于纸基平台的多重固相核酸杂交检测方法，该方法利用荧光共振能量转移（FRET）技术，以荧光素固定的量子点（QD）为供体。在纸的表面修饰咪唑基团，以覆盖在溶液中组装的两种类型的量子点-探针寡核苷酸缀合物。发绿光的QD（gQD）和发红光的QD（rQD）充当具有Cy 3和Alexa Fluor 647（A647）受体的供体。gQD/Cy 3 FRET对用作内标，而rQD/A647 FRET对用作检测通道，将对照和分析测试区组合在一个物理位置。染料标记的寡核苷酸靶标的杂交提供了来自受体染料的FRET敏化发射的接近性，其用作分析信号。杂交分析在ESTA格式提供了90 fmol的检测限和3.5 pmol的动态范围的上限。检测区阵列的使用被设计为提供与在一种类型的阵列设计上可以实现的分析品质因数相比，在荧光量子点的相对浓度方面改进的分析品质因数。杂交试验显示出良好的抵抗非特异性吸附的寡核苷酸。通过单核苷酸多态性（SNP）检测以50：1的对比率证明了双重杂交测定的选择性。此外，它表明，使用预先形成的量子点-探针寡核苷酸缀合物和考虑的相对数密度的两种类型的量子点-探针缀合物在双色测定格式是有利的，以最大限度地提高测定灵敏度和上限的动态范围。
A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.