Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

中文翻译：

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使用双乳滴增强基于 CRISPR/Cas12a 的核酸定量检测

由于其特异性、灵敏度和简单性，液滴微流体和 CRISPR/Cas12a 技术的配对为单分子水平的核酸检测和定量创建了强大的解决方案。然而，传统的油包水 (W/O) 单乳液 (SE) 液滴通常存在稳定性问题，影响测定结果的准确性和再现性。作为替代方案，水包油包水 (W/O/W) 双乳液 (DE) 液滴为液滴数字化验提供卓越的稳定性和均匀性。此外，与 SE 液滴不同，DE 液滴与市售流式细胞仪兼容，可进行高通量分析。尽管有这些优点，但没有研究证明 DE 液滴可用于基于 CRISPR 的核酸检测。在我们的研究中，我们进行了比较分析，以评估 SE 和 DE 液滴在基于 CRISPR/Cas12a 的人乳头瘤病毒 18 型 (HPV18) DNA 定量检测中的性能。我们通过检查不同温度和时间点孵育前后的大小变化、合并程度和内容相互作用来评估 SE 和 DE 的稳定性。通过将 DE 液滴与流式细胞术相结合，我们实现了基于 CRISPR/Cas12a 的目标 HPV18 DNA 的高通量和高精度定量。 DE 平台与 CRISPR/Cas12a 和流式细胞术技术配合使用，成为核酸生物标志物绝对定量的可靠工具。