An ultrasensitive enzyme-free electrochemical sandwich DNA biosensor is described for the detection of ssDNA oligonucleotides. A DNA sequence derived from the genom of Helicobacter pylori was selected as a model target DNA. The DNA assay was realized through catching target DNA on capture DNA immobilized gold electrode; then labeling the target DNA with reporter DNA (rpDNA) and initiator DNA (iDNA) co-modified gold nanoparticles (AuNPs). The high density of iDNAs serves as one of the amplification strategies. The iDNA triggers hybridization chain reaction (HCR) between two hairpins. This leads to the formation of a long dsDNA concatamer strand and represents one amplification strategy. The electrochemical probe [Ru(NH3)5L]2+, where L stands for 3-(2-phenanthren-9-ylvinyl)pyridine, intercalated into dsDNA chain. Multiple probe molecules intercalate into one dsDNA chain, serving as one amplification strategy. The electrode was subjected to differential pulse voltammetry for signal acquisition, and the oxidation peak current at −0.28 V was recorded. On each AuNP, 240 iDNA and 25 rpDNA molecules were immobilized. Successful execution of HCR at the DNA-modified AuNPs was confirmed by gel electrophoresis and hydrodynamic diameter measurements. Introduction of HCR significantly enhances the DNA detection signal intensity. The assay has two linear ranges of different slopes, one from 0.01 fM to 0.5 fM; and one from 1 fM to 100 fM. The detection limit is as low as 0.68 aM. Single mismatch DNA can be differentiated from the fully complementary DNA. Conceivably, this highly sensitive and selective assay provides a general method for detection of various kinds of DNA. Graphical abstract Schematic representation of the detection and the amplification principles of the electrochemical sandwich DNA assay. Purple curl: Captured DNA; Green curl: Reporter DNA; Orange curl: HCR initiator DNA; Yellow solid-circle: Gold nanoparticle; H1 and H2: Two hairpin DNA; [Ru(NH3)5L]2+: Signal probe. Schematic representation of the detection and the amplification principles of the electrochemical sandwich DNA assay. Purple curl: Captured DNA; Green curl: Reporter DNA; Orange curl: HCR initiator DNA; Yellow solid-circle: Gold nanoparticle; H1 and H2: Two hairpin DNA; [Ru(NH3)5L]2+: Signal probe.

中文翻译：

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基于杂交链反应和金纳米粒子的无酶电化学夹心 DNA 测定：在幽门螺杆菌 DNA 测定中的应用

描述了一种用于检测 ssDNA 寡核苷酸的超灵敏无酶电化学夹心 DNA 生物传感器。选择源自幽门螺杆菌基因组的DNA序列作为模型靶DNA。DNA检测是通过在捕获DNA固定化金电极上捕获目标DNA来实现的；然后用报告 DNA (rpDNA) 和起始 DNA (iDNA) 共修饰的金纳米粒子 (AuNPs) 标记目标 DNA。高密度的 iDNA 作为扩增策略之一。iDNA 触发两个发夹之间的杂交链反应 (HCR)。这导致形成长 dsDNA 多联体链并代表一种扩增策略。电化学探针 [Ru(NH3)5L]2+，其中 L 代表 3-(2-phenanthren-9-ylvinyl) 吡啶，插入 dsDNA 链。多个探针分子插入到一条 dsDNA 链中，作为一种扩增策略。对电极进行差分脉冲伏安法采集信号，记录-0.28 V的氧化峰电流。在每个 AuNP 上，固定了 240 个 iDNA 和 25 个 rpDNA 分子。凝胶电泳和流体动力学直径测量证实了在 DNA 修饰的 AuNP 上成功执行 HCR。HCR 的引入显着增强了 DNA 检测信号强度。该测定具有两个不同斜率的线性范围，一个从 0.01 fM 到 0.5 fM；一个从 0.01 fM 到 0.5 fM。一个从 1 fM 到 100 fM。检测限低至 0.68 aM。单个错配 DNA 可以与完全互补的 DNA 区分开来。可以想象，这种高度灵敏和选择性的检测提供了一种检测各种 DNA 的通用方法。图形摘要 电化学夹心 DNA 检测的检测和扩增原理的示意图。紫色卷曲：捕获的 DNA；绿色卷发：记者DNA；橙色卷曲：HCR 起始 DNA；黄色实心圆：金纳米颗粒；H1和H2：两个发夹DNA；[Ru(NH3)5L]2+：信号探针。电化学夹心 DNA 检测的检测和扩增原理的示意图。紫色卷曲：捕获的 DNA；绿色卷发：记者DNA；橙色卷曲：HCR 起始 DNA；黄色实心圆：金纳米颗粒；H1和H2：两个发夹DNA；[Ru(NH3)5L]2+：信号探针。图形摘要 电化学夹心 DNA 检测的检测和扩增原理的示意图。紫色卷曲：捕获的 DNA；绿色卷发：记者DNA；橙色卷曲：HCR 起始 DNA；黄色实心圆：金纳米颗粒；H1和H2：两个发夹DNA；[Ru(NH3)5L]2+：信号探针。电化学夹心 DNA 检测的检测和扩增原理的示意图。紫色卷曲：捕获的 DNA；绿色卷发：记者DNA；橙色卷曲：HCR 起始 DNA；黄色实心圆：金纳米颗粒；H1和H2：两个发夹DNA；[Ru(NH3)5L]2+：信号探针。图形摘要 电化学夹心 DNA 检测的检测和扩增原理的示意图。紫色卷曲：捕获的 DNA；绿色卷发：记者DNA；橙色卷曲：HCR 起始 DNA；黄色实心圆：金纳米颗粒；H1和H2：两个发夹DNA；[Ru(NH3)5L]2+：信号探针。电化学夹心 DNA 检测的检测和扩增原理的示意图。紫色卷曲：捕获的 DNA；绿色卷发：记者DNA；橙色卷曲：HCR 起始 DNA；黄色实心圆：金纳米颗粒；H1和H2：两个发夹DNA；[Ru(NH3)5L]2+：信号探针。电化学夹心 DNA 检测的检测和扩增原理的示意图。紫色卷曲：捕获的 DNA；绿色卷发：记者DNA；橙色卷曲：HCR 起始 DNA；黄色实心圆：金纳米颗粒；H1和H2：两个发夹DNA；[Ru(NH3)5L]2+：信号探针。电化学夹心 DNA 检测的检测和扩增原理的示意图。紫色卷曲：捕获的 DNA；绿色卷发：记者DNA；橙色卷曲：HCR 起始 DNA；黄色实心圆：金纳米颗粒；H1和H2：两个发夹DNA；[Ru(NH3)5L]2+：信号探针。