Empowering single-cell analysis with emerging active microfluidic devices

Understanding cellular heterogeneity is crucial for deciphering infection mechanisms, disease progression, and identifying novel biomarkers for personalized therapies, which provides valuable insights into biological processes and supports decision-making in biomedical settings. Microfluidics is a compact and lightweight system that enables efficient, high-throughput single-cell isolation and real-time analysis, making it the prevailing technology for single-cell analysis. Despite the advantages, microfluidic methods face several limitations, such as the need for off-chip pretreatment and post-processing, poor cell manipulation, and high cell damage, which hinder their broader application. Active microfluidics, which combines electrical, magnetic, acoustic, and optical technologies with microfluidic platforms, addresses some of these issues by creating a controlled microenvironment that enables precise, non-invasive, and high-throughput single-cell analysis. This review summarizes the latest advances in active microfluidics for applications, including nucleic acid, protein, cellular, and omic analysis. We also discuss the pros and cons of these technologies, offering guidance for researchers in selecting appropriate methods for desired single-cell analysis. Finally, we critically examine the current limitations of active microfluidic single-cell workflows and propose potential solutions to overcome these challenges.