AbstractA cascade nucleic acid amplification strategy is presented for fluorometric aptamer based determination of the model protein carcinoembryonic antigen (CEA). Amplification is accomplished by combining catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). In this assay, a specially designed single-stranded DNA containing the aptamer sequence (AS) specific for CEA is hybridized with an inhibitor strand (IS) to form a double-stranded DNA (IS@AS). In the presence of CEA, it will recognize and bind to the AS strand which causes the release of IS. By introducing two DNA hairpins (H1 and H2; these containing complementary sequences) CHA will be activated via the hybridization reactions of H1 and H2. This is accompanying by the formation of a double-stranded DNA (H1-H2) and the release of CEA@AS. The liberated CEA@AS further drives successive recycling of the CHA, thereby generating further copies of H1-H2. The resultant H1-H2 hybrids act as primers and trigger HCR with the help of other two DNA hairpins (H3 and H4) containing G-rich toehold at the 5′-terminus and 3′-terminus of H3 and H4, respectively. The fluorescent probe N-methyl mesoporphyrin IX (NMM) is finally intercalated into the G-rich domains of the long DNA nanostructures due to formation of G-quadruplex structures. This generates a fluorescent signal (best measured at excitation/emission wavelengths of 399/610 nm) that increases with the concentration of target (CEA). This aptamer-based fluorescence assay is highly sensitive and has a linear range that covers the 1 pg·mL−1 to 2 ng·mL−1 CEA concentration range, with a 0.3 pg·mL−1 detection limit. Graphical abstractBy integrating catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for effective signal enhancement, a novel cascade amplification strategy is presented to develop a sensitive and selective fluorescent method for the assay of the model protein carcinoembryonic antigen (CEA).

中文翻译：

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用于模型蛋白癌胚抗原灵敏荧光检测的催化发夹组装和杂交链反应的级联扩增策略

摘要提出了一种级联核酸扩增策略，用于基于荧光适体测定模型蛋白癌胚抗原 (CEA)。扩增是通过结合催化发夹组装 (CHA) 和杂交链反应 (HCR) 来完成的。在该测定中，一种专门设计的含有 CEA 特异性适体序列 (AS) 的单链 DNA 与抑制剂链 (IS) 杂交形成双链 DNA (IS@AS)。在 CEA 存在下，它会识别并结合导致 IS 释放的 AS 链。通过引入两个 DNA 发夹（H1 和 H2；它们包含互补序列），CHA 将通过 H1 和 H2 的杂交反应被激活。这伴随着双链 DNA (H1-H2) 的形成和 CEA@AS 的释放。解放的 CEA@AS 进一步推动了 CHA 的连续循环，从而产生了 H1-H2 的更多副本。所得的 H1-H2 杂交体作为引物，并在其他两个 DNA 发夹（H3 和 H4）的帮助下触发 HCR，这些发夹分别在 H3 和 H4 的 5'-末端和 3'-末端含有富含 G 的立足点。由于 G-四链体结构的形成，荧光探针 N-甲基中卟啉 IX (NMM) 最终插入到长 DNA 纳米结构的富含 G 的域中。这会产生一个荧光信号（最好在 399/610 nm 的激发/发射波长下测量），该信号随着目标浓度 (CEA) 的增加而增加。这种基于适体的荧光检测具有高度灵敏性，线性范围涵盖 1 pg·mL-1 至 2 ng·mL-1 CEA 浓度范围，检测限为 0.3 pg·mL-1。