Ultrahigh-throughput droplet microfluidic device for single-cell miRNA detection with isothermal amplification

• Published in: Lab on a chip Vol. 18; no. 13; pp. 1914 - 1920
• Main Authors: Guo, Song, Lin, Weikang Nicholas, Hu, Yuwei, Sun, Guoyun, Phan, Dinh-Tuan, Chen, Chia-Hung
• Format: Journal Article
• Language: English
• Published: England Royal Society of Chemistry 01.01.2018
• Subjects: Amplifiers; Assaying; Cascade chemical reactions; Deoxyribonucleic acid; DNA; Encapsulation; Flow cytometry; Flow measurement; Fluorescence; Gene expression; Gene sequencing; Polymerase chain reaction; Real time; Ribonucleic acid; RNA; Target detection; Thermal cycling
• ISSN: 1473-0197; 1473-0189
• DOI: 10.1039/c8lc00390d

Analysis of microRNA (miRNA), a pivotal primary regulator of fundamental cellular processes, at the single-cell level is essential to elucidate regulated gene expression precisely. Most single-cell gene sequencing methods use the polymerase chain reaction (PCR) to increase the concentration of the target gene for detection, thus requiring a barcoding process for cell identification and creating a challenge for real-time, large-scale screening of sequences in cells to rapidly profile physiological samples. In this study, a rapid, PCR-free, single-cell miRNA assay is developed from a continuous-flow microfluidic process employing a DNA hybridization chain reaction to amplify the target miRNA signal. Individual cells are encapsulated with DNA amplifiers in water-in-oil droplets and then lysed. The released target miRNA interacts with the DNA amplifiers to trigger hybridization reactions, producing fluorescence signals. Afterward, the target sequences are recycled to trigger a cyclic cascade reaction and significantly amplify the fluorescence signals without using PCR thermal cycling. Multiple DNA amplifiers with distinct fluorescence signals can be encapsulated simultaneously in a droplet to measure multiple miRNAs from a single cell simultaneously. Moreover, this process converts the lab bench PCR assay to a real-time droplet assay with the post-reaction fluorescence signal as a readout to allow flow cytometry-like continuous-flow measurement of sequences in a single cell with an ultrahigh throughput (300-500 cells per minute) for rapid biomedical identification.