Optimization of a reusable, DNA pseudoknot-based electrochemical sensor for sequence-specific DNA detection in blood serum.
Optimization of a reusable, DNA pseudoknot-based electrochemical sensor for sequence-specific DNA detection in blood serum.
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DOI：
10.1021/ac802011d
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发表时间：
2009-01-15
影响因子：
7.4
通讯作者：
Xiao, Yi
中科院分区：
文献类型：
--
作者：
Cash, Kevin J.;Heeger, Alan J.;Plaxco, Kevin W.;Xiao, Yi
我们详细描述了一种新的电化学DNA （E-DNA）传感平台，该平台基于电极结合DNA假结中靶诱导的构象变化。假结是一种包含两个茎环的DNA结构，其中第一个茎环形成第二个茎的一部分，在其3 ‘端被亚甲基蓝氧化还原标签修饰，并通过5 ’端共价连接到金电极上。在没有目标的情况下，假结探针的结构使氧化还原标签和电极之间的碰撞最小化，从而减少法拉第电流。目标结合破坏了假结结构，释放出一个灵活的单链元件，可以撞击电极并有效地转移电子。在本文中，我们报告进一步表征和优化这种新的E-DNA结构。我们发现在中间探针密度（~1.8 × 1013分子/cm2表观密度）下获得最佳信号，这可能代表了高探针密度下的位阻和静电阻塞与低密度下假结探针转移引起的背景电流增加之间的平衡。我们还发现，最佳的3 '茎长，似乎是7个碱基对，代表了假结结构稳定性和目标亲和力之间的平衡。最后，由poly(a)组成的3 '环比等效的poly(T)环表现出更好的错配判别，但代价是降低了增益。在这个参数空间上的优化显著改善了基于假结的E-DNA结构的信号传导，即使在复杂的、多组分的样品（如血清）中受到挑战，也能敏感和特异性地检测DNA靶标。
We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem’s loop forms part of the second stem, is modified with a methylene blue redox tag at its 3′ terminus and covalently attached to a gold electrode via the 5′ terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density (~1.8 × 1013 molecules/cm2 apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3′ stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3′ loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum.
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影响因子：
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Ricci, Francesco;Lai, Rebecca Y.;Plaxco, Kevin W.
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