Hybridization efficiency of molecular beacons bound to gold nanowires: effect of surface coverage and target length.
Hybridization efficiency of molecular beacons bound to gold nanowires: effect of surface coverage and target length.
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DOI：
10.1021/la1031703
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发表时间：
2010-12-07
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;Keating CD
设计为采用特定二级结构的表面结合核酸探针在一系列生物传感应用中变得越来越重要，但与传统的单链探针相比仍然没有得到很好的表征，传统的单链探针通常被设计为避免二级结构。我们报告了表面固定的发夹 DNA 探针的杂交效率。我们的探针是分子信标，带有 3' 染料部分和 5' 硫醇，用于附着在金纳米线上，金纳米线既充当探针附着的支架又充当猝灭剂。杂交效率取决于探针表面覆盖度，在 (1−2) × 1012 个探针/cm2 的中间覆盖度下杂交效率最高可达 ∼90%，在较高或较低的覆盖度下降至 ≤20%。荧光强度不随结合的目标分子的数量而变化，并且尽管每平方厘米的结合目标分子较低，但在高探针覆盖率下荧光强度最高。用短硫醇化低聚乙二醇间隔基回填可提高低发夹探针覆盖率（∼(3−4) × 1011 个探针/cm2）下的杂交效率，但在较高探针覆盖率（1 × 1012/cm2）下则不会提高杂交效率。我们还通过在互补靶序列的 3' 或 5' 末端添加多达 50 个非杂交核苷酸来评估靶长度的影响。与添加到 5' 端的核苷酸相比，互补靶序列 3' 端（即靠近纳米线表面的末端）上的附加核苷酸对杂交效率的影响要大得多。这项工作为设计传感器提供了指导，其中设计为采用二级结构的表面结合探针用于检测溶液中的目标序列。
Surface-bound nucleic acid probes designed to adopt specific secondary structures are becoming increasingly important in a range of biosensing applications but remain less well characterized than traditional single-stranded probes, which are typically designed to avoid secondary structure. We report the hybridization efficiency for surface-immobilized hairpin DNA probes. Our probes are molecular beacons, carrying a 3′ dye moiety and a 5′ thiol for attachment to gold nanowires, which serve as both scaffolds for probe attachment and quenchers. Hybridization efficiency was dependent on probe surface coverage, reaching a maximum of ∼90% at intermediate coverages of (1−2) × 1012 probes/cm2 and dropping to ≤20% at higher or lower coverages. Fluorescence intensity did not track with the number of target molecules bound, and was highest for high probe coverage despite the lower bound targets per square centimeter. Backfilling with short thiolated oligoethylene glycol spacers increased hybridization efficiency at low hairpin probe coverages (∼(3−4) × 1011 probes/cm2), but not at higher probe coverages (1 × 1012/cm2). We also evaluated the effect of target length by adding up to 50 nonhybridizing nucleotides to the 3′ or 5′ end of the complementary target sequence. Additional nucleotides on the 3′ end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on hybridization efficiency as compared to nucleotides added to the 5′ end. This work provides guidance in designing sensors in which surface-bound probes designed to adopt secondary structures are used to detect target sequences from solution.
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影响因子：
15
作者：
Jayagopal, Ashwath;Halfpenny, Kristin C.;Perez, Jonas W.;Wright, David W.
通讯作者：
Wright, David W.
影响因子：
15
作者：
Du, H;Disney, MD;Krauss, TD
通讯作者：
Krauss, TD
影响因子：
29.4
作者：
Keating, CD;Natan, MJ
通讯作者：
Natan, MJ
影响因子：
46.9
作者：
Dubertret, B;Calame, M;Libchaber, AJ
通讯作者：
Libchaber, AJ
DOI：
10.1073/pnas.1633515100
发表时间：
2003-08-05
影响因子：
11.1
作者：
Fan, CH;Plaxco, KW;Heeger, AJ
通讯作者：
Heeger, AJ