Jet Thomas, Kieffer Coline, Rondelez Yannick, Taly Valérie, Gines Guillaume
Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Cité, MEPPOT/Equipe labellisée Ligue Nationale Contre le Cancer/INSERM UMR-S1138, CNRS SNC5096, Paris F-75006, France.
Gulliver Laboratory, UMR7083 CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, Paris 75005, France.
J Am Chem Soc. 2025 Jul 23;147(29):25705-25714. doi: 10.1021/jacs.5c07214. Epub 2025 Jul 11.
The sensitive and accurate detection of panels of microRNA molecules is critical to enable their use as disease biomarkers. While microarrays and next-generation sequencing allow comprehensive miRNA profiling, they generally suffer from low accuracy and sensitivity. Conversely, digital bioassays such as digital PCR provide an absolute quantification with unrivaled sensitivity, although with limited multiplexing capabilities. In this work, we describe a novel microRNA profiling procedure, termed Digiplex, that combines the multiplexing power of a DNA-grafted suspension array with the accuracy of a digital flow cytometry readout. microRNAs are first captured on fluorescently encoded DNA-grafted particle populations following a Poisson distribution. The particles are subsequently isolated in microfluidic droplets, where single captured miRNA molecules trigger an isothermal exponential amplification that ultimately activates a fluorescent probe on the particle surface. Flow cytometry analysis yields the ratio of positive to negative particles for each targeted microRNA, allowing the reconstruction of the multiplex concentration profile in one go. After optimizing the workflow on a single microRNA model, we successfully developed a 10-plex assay with femtomolar sensitivity. The Digiplex method was finally validated on total RNA samples and benchmarked against droplet digital PCR.
对微小RNA分子组进行灵敏且准确的检测对于将其用作疾病生物标志物至关重要。虽然微阵列和下一代测序能够实现全面的miRNA分析，但它们通常存在准确性和灵敏度较低的问题。相反，诸如数字PCR之类的数字生物测定法虽具有无与伦比的灵敏度可进行绝对定量，但多重分析能力有限。在这项工作中，我们描述了一种名为Digiplex的新型miRNA分析程序，它将DNA接枝悬浮阵列的多重分析能力与数字流式细胞术读数的准确性相结合。首先，按照泊松分布将微小RNA捕获在荧光编码的DNA接枝颗粒群体上。随后，将这些颗粒分离到微流控液滴中，单个捕获的miRNA分子在其中触发等温指数扩增，最终激活颗粒表面的荧光探针。流式细胞术分析得出每个靶向微小RNA的阳性与阴性颗粒的比例，从而一次性重建多重浓度分布图。在单个微小RNA模型上优化工作流程后，我们成功开发了一种具有飞摩尔灵敏度的10重分析方法。最后，在总RNA样本上对Digiplex方法进行了验证，并与液滴数字PCR进行了比较。
