Sensitivity and specificity of metal surface-immobilized "molecular beacon" biosensors
Sensitivity and specificity of metal surface-immobilized "molecular beacon" biosensors
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DOI：
10.1021/ja042482a
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发表时间：
2005-06-01
影响因子：
15
通讯作者：
Krauss, TD
中科院分区：
文献类型：
--
作者：
Du, H;Strohsahl, CM;Krauss, TD
DNA寡核苷酸微阵列图案化和DNA发夹作为溶液中识别寡核苷酸的灵敏探针的发展，对基础生物学研究和临床应用产生了巨大的影响。我们将这两种方法结合起来，基于固定在金属表面的DNA发夹的荧光不猝灭，开发了可阵列和无标记的生物传感器。研究了这些传感器的热力学和动力学响应，以及影响杂交效率的重要因素。杂交效率对发夹二级结构以及DNA发夹在底物上的表面分布很敏感。发夹茎中使用的碱基的同一性以及整个环的长度显着影响灵敏度和选择性。表面固定的发夹在相同的检测条件下(严格程度不变)以高灵敏度(超过一个数量级的信号差异)区分具有单个碱基对错配的两个序列。这代表着与其他基于微阵列的技术相比有了显著的进步。
The separate developments of microarray patterning of DNA oligonucleotides, and of DNA hairpins as sensitive probes for oligonucleotide identification in solution, have had a tremendous impact on basic biological research and clinical applications. We have combined these two approaches to develop arrayable and label-free biological sensors based on fluorescence unquenching of DNA hairpins immobilized on metal surfaces. The thermodynamic and kinetic response of these sensors, and the factors important in hybridization efficiency, were investigated. Hybridization efficiency was found to be sensitive to hairpin secondary structure, as well as to the surface distribution of DNA hairpins on the substrate. The identity of the bases used in the hairpin stem as well as the overall loop length significantly affected sensitivity and selectivity. Surface-immobilized hairpins discriminated between two sequences with a single base-pair mismatch with high sensitivity (over an order of magnitude difference in signal) under identical assay conditions (no change in stringency). This represents a significant improvement over other microarray-based techniques.