Digital droplet technology has emerged as a powerful new tool for biomarker analysis. Temperature cycling, enzymes, and off-chip processes are, nevertheless, always required. Herein, we constructed a digital droplet auto-catalytic hairpin assembly (ddaCHA) microfluidic system to achieve digital quantification of single-molecule microRNA (miRNA). The designed continuous chip integrates droplet generation, incubation, and fluorescence imaging on the chip, avoiding the requirement for extra droplet re-collection and heating operations. Clearly, the digital readout was obtained by partitioning miRNA into many individual pL-sized small droplets in which the target molecule is either present (“positive”) or absent (“negative”). Importantly, the suggested enzyme-free auto-catalytic hairpin assembly (aCHA) in droplets successfully mitigated the effects of the external environment and thermal cycling on droplets, and its reaction rate is significantly superior to that of traditional CHA. We got excellent sensitivity with a linear correlation from 1 pM to 10 nM and a detection limit of 0.34 pM in the fluorescence spectrum section, as well as high selectivity to other miRNAs. Furthermore, the minimum target concentration could be reduced to 10 fM based on the high-throughput tracking computation of fluorescent droplets with a self-developed Python script, and the fluorescence intensity distribution agreed well with the theoretical value, demonstrating that it is feasible to detect miRNA efficiently and accurately, which has great potential applications in clinical diagnostics and biochemical research.

中文翻译：

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具有高通量荧光成像和液滴跟踪计算的一步数字液滴自动催化核酸扩增

数字液滴技术已成为一种强大的生物标志物分析新工具。然而，始终需要温度循环、酶和芯片外工艺。在此，我们构建了数字液滴自催化发夹组装（ddaCHA）微流控系统，以实现单分子微RNA（miRNA）的数字量化。设计的连续芯片在芯片上集成了液滴生成、孵化和荧光成像，避免了额外的液滴重新收集和加热操作的要求。显然，数字读数是通过将 miRNA 分成许多单独的 pL 大小的小液滴获得的，其中目标分子存在（“阳性”）或不存在（“阴性”）。重要的，建议的液滴中无酶自催化发夹组装（aCHA）成功地减轻了外部环境和热循环对液滴的影响，其反应速率明显优于传统的CHA。我们获得了出色的灵敏度，线性相关性从 1 pM 到 10 nM，荧光光谱部分的检测限为 0.34 pM，并且对其他 miRNA 具有高选择性。此外，基于自研Python脚本对荧光液滴的高通量跟踪计算，最小目标浓度可降低至10 fM，荧光强度分布与理论值吻合较好，证明检测是可行的。 miRNA高效准确，