2.10. Liquid AFM Imaging

AFM images were acquired on a Cypher ES AFM (Asylum Research) using PNP-DB tips with a nominal spring constant of ≈0.5 N/m and drive frequency of ≈67 kHz. For sample preparation, mica was first treated in an aminopropyl silatrane (APS) solution (67 µM) for 30 min to functionalize the surface and then washed thoroughly with ultrapure water and PBS. The samples were then deposited onto the surface (50 µL droplet, all samples had a concentration of 10 µg/mL) and allowed to bind for 20 min at room temperature. The surface was then washed again with PBS and placed into the AFM for imaging. Care was taken to ensure the samples were never allowed to dry. All images were taken at room temperature in standard tapping mode with a resolution of 256 × 256 pixels. Additionally, blueDrive (Asylum Research, Oxford Instruments) photothermal excitation was utilized to ensure high quality imaging in liquid conditions.

Images obtained were processed using the “Particle Analysis” function in the Asylum Research AFM software, which yielded heights for all particles in the images. These values were then grouped by sample and exported into MATLAB (R2020b). A one-way analysis of variance (ANOVA) was then performed on the grouped data to determine significant differences.