Nanowizardtm

All images were collected using an AFM (Nanowizard^TM, JPK instruments, Berlin, Germany) installed on an inverted optical microscope (Nikon Corporation, Tokyo, Japan). The AFM probes used were oxidize-sharpened silicon nitride probes (OMCL-TR400PB-1, Olympus, Tokyo, Japan) with a spring constant of 0.02 N/m. Ten microliters of samples treated with or without gramicidin A or Ca²⁺ ions at the designed concentration were placed on a cleaved mica disc (Ted Pella Inc., Redding, CA, USA) for 20–40 min. The mica disc was then rinsed twice with distilled water and dried for 24 h. The dried samples were imaged in an AFM operating in a contact mode. Images were acquired at a scanning rate of 1–2 Hz and a resolution of 512 × 512 pixels. Image processing and analysis were performed using SPM software v. 3.16 (Nanowizard^TM, JPK instruments, Berlin, Germany, Germany).

The morphology of p116-assembled particles was visualized using a transmission electron microscope (H-7500, Hitachi, Tokyo, Japan) with an accelerating voltage of 100 KeV. 10 μL of the p116-assembled particle-containing solution was placed onto a carbon-coated 300-mesh copper grid (PELCO, Ted Pella, Inc., Redding, CA, USA) and stayed for 1 min. The excess solution was wicked dry, and the sample was stained with 10 μL 1% phosphotungstic acid for 30 s (negative staining). The excess solution was again wicked dry, and the grid was allowed to air dry before TEM imaging. For AFM imaging, 50 μL the sample containing p116-assembled particles was dropped onto a freshly cleaved mica sheet and allowed to stay for 40 min for adsorption. The sheet was washed twice with Milli-Q water and air-dried for 48 h in the dry cabinet before AFM imaging. AFM measurements were performed with an atomic force microscope (NanoWizard^TM, JPK Instruments, Berlin, Germany). The AFM probe used was 200 mm-long gold-coated cantilevers with oxide sharpened Si₃N₄ tips (Olympus, Tokyo, Japan). The spring constant for the cantilevers was 0.02 nN/nm. The scan rate was 1 Hz.