Du Hui, Strohsahl Christopher M, Camera James, Miller Benjamin L, Krauss Todd D
Department of Chemistry, and The Center for Future Health, University of Rochester, Rochester, New York, USA.
J Am Chem Soc. 2005 Jun 1;127(21):7932-40. doi: 10.1021/ja042482a.
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.
DNA寡核苷酸的微阵列图案化以及作为溶液中寡核苷酸识别灵敏探针的DNA发夹的独立发展，对基础生物学研究和临床应用产生了巨大影响。我们将这两种方法结合起来，开发了基于固定在金属表面的DNA发夹荧光去淬灭的可阵列化且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，以及对杂交效率重要的因素。发现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敏感。发夹茎中使用的碱基身份以及整个环的长度显著影响灵敏度和选择性。在相同的检测条件下（严格度不变），表面固定的发夹能够以高灵敏度区分两个具有单个碱基对错配的序列（信号差异超过一个数量级）。这相对于其他基于微阵列的技术有了显著改进。
