Optimized linkage and quenching strategies for quantum dot molecular beacons
Optimized linkage and quenching strategies for quantum dot molecular beacons
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DOI：
10.1016/j.mcp.2006.09.001
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发表时间：
2007-04-01
影响因子：
3.3
通讯作者：
Batt, Carl A.
中科院分区：
文献类型：
--
作者：
Cady, Nathaniel C.;Strickland, Aaron D.;Batt, Carl A.
量子点（QD）分子信标被用于序列特异性DNA检测。在基于杂交的检测中，对多种连接策略和荧光猝灭剂的效果进行了比较。为了比较连接策略，使用共价酰胺连接和链霉亲和素 - 生物素结合将半导体量子点连接到分子信标DNA上。当与200皮摩尔的靶DNA杂交时，酰胺连接的信标比链霉亲和素连接的信标荧光增强幅度高57%。然而，两种连接方法的分子信标特异性相似。两种量子点分子信标与非互补靶DNA杂交时的荧光强度比与互补DNA杂交时大约低50%。还评估了不同猝灭剂部分的效果。当与互补靶标杂交时，爱荷华黑（Iowa Black）和1.4纳米纳米金猝灭的分子信标比达比克（dabcyl）猝灭的信标荧光增强幅度大约高2倍。通过与非互补DNA的杂交检测也证实了对靶DNA的特异性。为了深入了解量子点分子信标和所使用的连接策略之间的差异，测量了每种的流体动力学半径。这些测量结果表明，链霉亲和素量子点（13.5纳米）比羧基量子点（7纳米）的半径更大，这可能对量子点分子信标的基于荧光共振能量转移（FRET）的猝灭产生负面影响。这些数据概述了选择合适的连接方法和猝灭剂部分对于构建高质量量子点分子信标的重要性。（c）2006爱思唯尔有限公司。保留所有权利。
Quantum dot (QD) molecular beacons were explored for sequence-specific DNA detection. The effectiveness of multiple linkage strategies and fluorescence quenchers were compared in hybridization-based assays. To compare linkage strategies, covalent amide linkage and streptavidin-biotin binding were used to link semiconductor QDs to molecular beacon DNA. Amide-linked beacons showed a 57% greater fluorescence increase than streptavidin-linked beacons when hybridized to 200 pmol of target DNA. The specificity of the molecular beacons, however, was similar for both linkage methods. Hybridization of both QD molecular beacons with non-complementary target DNA resulted in approximately 50% lower fluorescence intensity than hybridization with complementary DNA. The effectiveness of different quencher moieties was also evaluated. Iowa Black and 1.4nm Nanogold-quenched molecular beacons exhibited approximately 2-fold greater fluorescence increases than dabcyl-quenched beacons when hybridized to complementary target. Specificity for target DNA was also confirmed through hybridization assays with non-complementary DNA. To provide insight into differences between the QD molecular beacons and the linkage strategies used, the hydrodynamic radius of each was measured. These measurements indicated that the larger radius of the streptavidin QDs (13.5 nm) than the carboxyl QDs (7 nm) could have a negative effect on FRET-based quenching for QD molecular beacons. These data outline the importance of choosing proper linkage methods and quencher moieties for creating high-quality QD molecular beacons. (c) 2006 Elsevier Ltd. All rights reserved.