Production of Stable Monodisperse Phospholipid-coated Microbubbles at Room Temperature Using a Microfluidic Flow-focusing Device

Monodisperse microbubbles have demonstrated significant potential in enhancing the efficacy of ultrasound contrast agents for both imaging and therapeutic applications. These microbubbles can be directly produced using a microfluidic flow-focusing method, which allows precise control over their size. However, preventing coalescence during production remains a critical challenge. While elevated production temperatures (e.g., 55 °C) can suppress coalescence, such conditions complicate microfluidic device design and may be incompatible with targeting agents and drug conjugates. This protocol outlines a method for producing monodisperse phospholipid-coated microbubbles at room temperature, with the microbubbles' stability and monodispersity maintained for at least 7 days. The protocol also describes the fabrication of reusable PDMS microfluidic chips and preparation of the phospholipid coating formulation containing the surfactant Pluronic F68. Our findings show that the microbubbles produced using this method maintain their size and stability over 7 days, supporting their future applicability in clinical settings where stable and controllable ultrasound contrast agents are essential.