Molecular beacon-metal nanowire interface: effect of probe sequence and surface coverage on sensor performance.
Molecular beacon-metal nanowire interface: effect of probe sequence and surface coverage on sensor performance.
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DOI：
10.1021/la703854x
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发表时间：
2008-08-19
期刊：
影响因子：
--
通讯作者：
Keating CD
中科院分区：
文献类型：
--
作者：
Cederquist KB;Stoermer Golightly R;Keating CD
我们报告了表面覆盖度和序列对5′巯基化、3′荧光团标记的DNA发夹探针与Au/Ag条纹（“条形码”）金属纳米线结合的性能的影响。通过改变探针浓度、缓冲液离子强度以及在探针组装到纳米线表面期间通过添加短的羟基封端的烷醇稀释剂分子来控制覆盖。用ω-羟基烷醇稀释表面结合的探针，这是表面结合的DNA文献中普遍接受的做法，与没有羟基烷醇稀释剂的类似探针覆盖率相比，没有明显改善传感器性能;这一发现强调了这里使用的分子信标探针与更传统的非荧光无规卷曲探针之间的差异。我们发现，约1012分子/cm 2的中间探针覆盖度给出了靶序列的存在和不存在之间的最佳区分。因为我们感兴趣的多重测定，我们还比较了几个信标探针序列具有不同的稳定性，在溶液中的二级结构形成，我们发现，探针表面覆盖和传感器的性能不同的探针序列。当五种不同的分子信标探针（每种都与条形码纳米线结合）用于对应于病毒核酸序列的靶寡核苷酸的多重免洗涤测定时，探针性能的这些差异并不妨碍准确的靶鉴定。我们预期，这里描述的发现也将与涉及固定在金属表面上的分子信标或其他结构化核酸探针的其他应用相关。
We report the effect of surface coverage and sequence on the performance of 5′ thiolated, 3′ fluorophore-labeled DNA hairpin probes bound to Au/Ag striped (“barcoded”) metal nanowires. Coverage was controlled by varying probe concentration, buffer ionic strength, and by addition of short hydroxy-terminated alkanethiol diluent molecules during probe assembly onto the nanowire surface. Surface dilution of the surface-bound probes with aω-hydroxyl alkanethiol, a commonly accepted practice in the surface-bound DNA literature, did not appreciably improve sensor performance as compared to similar probe coverages without hydroxyalkanethiol diluents; this finding underscores the differences between the molecular beacon probes used here and more traditional nonfluorescent, random coil probes. We found that intermediate probe coverage of ~1012 molecules/cm2 gave the best discrimination between presence and absence of a target sequence. Because we are interested in multiplexed assays, we also compared several beacon probe sequences having different stabilities for secondary structure formation in solution; we found that both probe surface coverage and sensor performance varied for different probe sequences. When five different molecular beacon probes, each bound to barcoded nanowires, were used in a multiplexed, wash-free assay for target oligonucleotides corresponding to viral nucleic acid sequences, these differences in probe performance did not prevent accurate target identification. We anticipate that the findings described here will also be relevant to other applications involving molecular beacons or other structured nucleic acid probes immobilized on metal surfaces.
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