Biophysical restrictions regulate protein diffusion, nucleus deformation, and cell migration, which are all universal and important processes for cells to perform their biological functions. However, current technologies addressing these multiscale questions are extremely limited. Herein, through two-photon polymerization (TPP), we present the precise, low-cost, and multiscale microstructures (micro-fences) as a versatile investigating platform. With nanometer-scale printing resolution and multiscale scanning capacity, TPP is capable of generating micro-fences with sizes of 0.5–1000 μm. These micro-fences are utilized as biophysical restrictions to determine the fluidity of supported lipid bilayers (SLB), to investigate the restricted diffusion of Src family kinase protein Lck on SLB, and also to reveal the mechanical bending of cell nucleus and T cell climbing ability. Taken together, the proposed versatile and low-cost micro-fences have great potential in probing the restricted dynamics of molecules, organelles, and cells to understand the basics of physical biology. Graphic abstract
Supported lipid bilayers (SLBs), two-dimensional lipid films formed on a solid-supporting substrate, serve as models for biomembranes and exhibit remarkable potential in chemistry, biology, and medicine. However, preparing SLBs with highly negatively charged contents on the negatively charged surface by overcoming electrostatic repulsion remains a challenge. Here, a creative bicelle-mediated and divalent cation-free SLB preparation method with the assistance of phosphate-buffered saline (PBS) solution was proposed, which can form the SLBs containing 50% DOPS or 30% CL on the silica surface monitored by a quartz crystal microbalance with dissipation (QCM-D). Results of molecular dynamics (MD) simulation indicate that electrostatic repulsion can be overcome by the increased number of hydrogen bonds caused by the adsorption of dihydrogen phosphate ions onto the headgroups of lipids. In addition, the negatively charged SLB formation was identified to be a three-step kinetic process, which differs from a two-step mechanism in the case of amphoteric SLB. The extra kinetic step can be attributed to the reduction in the number of intermolecular hydrogen bonds and the ordering of water molecules in the hydration layer. This investigation resolves the challenge of fabricating SLB over negatively charged surfaces and offers a fresh perspective on the SLB assembly methodology.
危险品化学品经营许可证（带存储）