Analysis of single-cell microRNA is essential to reveal cell heterogeneity at the genetic level. It raises a high demand for single-cell analytical methods because single-cell microRNA sequences are highly similar and small in size and feature low-level expression. Herein, SERS and fluorescence imaging technology were introduced into a microfluidic droplet platform to realize direct in situ, nondestructive, and highly sensitive detection of a small number of microRNA-21 (miR-21) in a single intact living cell. A multifunctional plasmonic nanoprobe was designed by decorating a gold nanoparticle with fluorescent dye (ROX)-labeled probe DNA and capture DNA strands. The dual-signal switching of fluorescence turn-off and SERS turn-on of ROX in response to miR-21 achieves highly sensitive and reliable detection of miR-21 in a single cell. The turn-on of SERS signal with a zero background guarantees the sensitivity of the detection. The fluorescence-SERS simultaneous response strategy was able to mutually corroborate the test results, improving the reliability of determining low-level expression of miR-21. SERS combined with encapsulation of microdroplets provides a feasible way to conduct in situ, nondestructive determination of miR-21 secreted by single cells, avoiding cell lysis and tedious time-consuming steps of miR-21 isolation. As a result, the miR-21 expressed by various types of single cells was investigated by fluorescence imaging and the cellular heterogeneity in miR-21 expression was evaluated accurately and quantitatively by SERS. This research would provide important reference information for understanding the effects of miRNAs on cancer diseases at the single-cell level.

中文翻译：

---

荧光和 SERS 双响应微流体液滴平台揭示单个活细胞中 MicroRNA-21 的表达

单细胞 microRNA 的分析对于在遗传水平上揭示细胞异质性至关重要。由于单细胞 microRNA 序列高度相似、体积小、表达水平低，因此对单细胞分析方法提出了很高的要求。在此，将SERS和荧光成像技术引入微流控液滴平台，实现直接原位、无损和高灵敏度检测单个完整活细胞中的少量 microRNA-21 (miR-21)。通过用荧光染料 (ROX) 标记的探针 DNA 装饰金纳米粒子并捕获 DNA 链，设计了一种多功能等离子体纳米探针。ROX响应miR-21的荧光关闭和SERS开启的双信号切换实现了单细胞中miR-21的高灵敏度和可靠检测。以零背景开启 SERS 信号保证了检测的灵敏度。荧光-SERS同时响应策略能够相互印证测试结果，提高了确定miR-21低水平表达的可靠性。SERS 与微滴封装相结合，为原位进行提供了一种可行的方法, 无损检测单细胞分泌的 miR-21，避免了细胞裂解和 miR-21 分离的繁琐耗时的步骤。结果，通过荧光成像研究了由各种类型的单细胞表达的miR-21，并通过SERS准确定量地评估了miR-21表达的细胞异质性。该研究将为了解miRNAs在单细胞水平上对癌症疾病的影响提供重要的参考信息。