As an amplification tool, DNA walker nanomachine exhibits promising potential in the fields of biosensing. To further improve amplification efficiency of DNA walker, a number of cascade amplification systems have been developed, but they still suffer from separated steps and long reaction time. Herein, by combining entropy-driven catalytic (EDC) amplification with DNAzyme reaction, a highly integrated cascade three-dimensional (3D) DNA walker is constructed, which allows the autonomous DNA circuit to be completed in one step. Further combined with element tag and inductively coupled plasma mass spectrometry (ICP-MS) detection, the cascade walker can be used for quantification of miRNA-21. Specifically, the reactant of EDC (WLT complex) and the substrate of DNAzyme (Substrate-Tb) are both fixed on magnetic beads (MBs) to assemble the track of walker. Once cascade DNA walker is triggered by target miRNA, EDC amplification runs first and keeps circulating to liberate many DNAzyme strands. DNAzyme can repeatedly cleave Substrate-Tb with autonomously walking along the substrate on MBs. As a result, a great number of fragments of Substrate-Tb, as output signal, are generated within 1 h, resulting in elemental signal response in ICP-MS and reflecting the amount of miRNA-21. A good linear relationship was obtained with miRNA-21 concentration in the range of 0.02−1 nmol L⁻¹, and the detection limit of 5 pmol L⁻¹ was achieved. The recoveries of miRNA-21 in human serum were 92.0–107 %. The developed method provided a novel idea for constructing highly integrated and efficient cascade DNA walker with ICP-MS strategy for bioanalysis.

作为一种放大工具，DNA walker 纳米机器在生物传感领域展现出巨大的潜力。为了进一步提高DNA walker的扩增效率，已经开发了许多级联扩增系统，但它们仍然存在步骤分离和反应时间长的问题。在此，通过将熵驱动催化 (EDC) 扩增与 DNAzyme 反应相结合，构建了高度集成的级联三维 (3D) DNA walker，从而一步完成自主 DNA 回路。进一步结合元素标签和电感耦合等离子体质谱 (ICP-MS) 检测，级联步行器可用于 miRNA-21 的定量。具体来说，EDC的反应物（WLT复合物）和DNAzyme的底物（底物-Tb）都固定在磁珠（MBs）上以组装步行者的轨迹。一旦级联 DNA walker 被目标 miRNA 触发，EDC 扩增首先运行并保持循环以释放许多 DNAzyme 链。DNAzyme 可以通过在 MB 上自主地沿着底物行走来重复切割底物-Tb。结果，在 1 h 内产生了大量的 Substrate-Tb 片段作为输出信号，导致 ICP-MS 中的元素信号响应并反映了 miRNA-21 的数量。miRNA-21 浓度在 0.02-1 nmol L 范围内获得了良好的线性关系 DNAzyme 可以通过在 MB 上自主地沿着底物行走来重复切割底物-Tb。结果，在 1 h 内产生了大量的 Substrate-Tb 片段作为输出信号，导致 ICP-MS 中的元素信号响应并反映了 miRNA-21 的数量。miRNA-21 浓度在 0.02-1 nmol L 范围内获得了良好的线性关系 DNAzyme 可以通过在 MB 上自主地沿着底物行走来重复切割底物-Tb。结果，在 1 h 内产生了大量的 Substrate-Tb 片段作为输出信号，导致 ICP-MS 中的元素信号响应并反映了 miRNA-21 的数量。miRNA-21 浓度在 0.02-1 nmol L 范围内获得了良好的线性关系^-1，并且达到了 5 pmol L ^-1的检测限。人血清中 miRNA-21 的回收率为 92.0-107%。所开发的方法为构建高度集成且高效的级联 DNA Walker 与 ICP-MS 策略提供了一种新的思路进行生物分析。