Total internal reflection fluorescence microscopy to study sheet front growth in phospholipid supported lipid membrane formation

Abstract

Supported lipid bilayer (SLB) based biosensors possess biomedical applications such as in rapid detection of antigens and cytochromes. It is generally believed that the SLB can be formed by adsorbing and spontaneously rupturing vesicles on substrate. Recent findings highlight the importance of investigating the adsorption and rupture of individual vesicles during the SLB formation. Here, we use total internal reflection fluorescence microscopy (TIRFM) to characterize the spatiotemporal kinetics of the front spreading at patch boundary. Owing to the mixture of labeled and unlabeled vesicles individual vesicle or patch on the surface can be identified. The TIRFM is employed to investigate the adsorption, rupture of vesicles, and spreading of the patch front. Combining quartz crystal microbalance with dissipation monitoring (QCM-D) and TIRFM characterizations, we find that the size of vesicle has a significant effect on the front spreading at the patch boundary. Quantification of the number of patches and patches area displays that smaller vesicles are more prone to the formation of patches. The front spreading at the patch boundary is analyzed quantitatively using the average front growth velocity (<inline-formula><tex-math id="M3">\begin{document}$ {v}_{\rm afv} $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="16-20220309_M3.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="16-20220309_M3.png"/></alternatives></inline-formula>), which indicates that the <inline-formula><tex-math id="M4">\begin{document}$ {v}_{\rm afv} $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="16-20220309_M4.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="16-20220309_M4.png"/></alternatives></inline-formula>of 40-nm vesicles is one order of magnitude larger than that of the 112 nm vesicles. Both theoretical analysis and experimental observation show that the smaller vesicles can attain the higher concentration on the surface (<i>C</i>) and high diffusivity in the medium. The global growth theoretical model (GGM) presents that for the patches with the same surface area and vesicle exposure time, the growth of the patch depends on <i>C</i> and lipid loss percentage during the vesicle rupture. The calculated lipid loss of the smaller vesicles is slightly higher than that of the larger vesicles, while <i>C</i> plays a dominating role in determining the disparity of the patch growth between the different vesicles. This study promotes the understanding of the growth mechanism of patches on the surface. It demonstates the critcial role of the supply of vesicles in this process and provides an enlightenment for investigating the reassembly of lipids on a nanoscale.