Twenty years of microfluidic probes and open-space microfluidics: from origins to emerging directions.

Microfluidic probes (MFPs) are an emerging class of open microfluidic devices that use hydrodynamic flow confinement (HFC) to enable precise, contact-free delivery, and removal of fluids on biological surfaces. Unlike closed-channel microfluidics, MFPs operate in open environments, allowing localized chemical and biological interactions with high spatial and temporal resolution. Since their introduction in 2005, MFPs have advanced through major innovations, including multipolar flow designs, vertical configurations, 3D printing, and structural enhancements such as herringbone micromixers. This review presents a comprehensive overview of MFP technologies, covering core physical principles, flow dynamics, operating modes, and the influence of geometric and hydrodynamic design. We examine fabrication techniques such as photolithography, soft lithography, and 3D printing, highlighting their trade-offs in precision, scalability, and cost. We also explore biological applications of MFPs, including tissue assays, cellular manipulation, molecular patterning, and single-cell biopsy. Emerging integrations with heating, dielectrophoresis, and real-time feedback are expanding the utility of MFPs for adaptive high-throughput workflows. By tracing two decades of development, this review positions MFPs as transformative tools in open-space microfluidics and outlines opportunities for future progress.