AFM imaging of extracellular ice nucleators

Ice nucleators are substances that can initiate nucleation of pure water at and above -10 °C. Some plant pathogens possess a gene which encodes for a protein that acts as an ice nucleator, activity of which is enhanced when it is combined with the sugar and lipid components from the cell membrane. This matter retains its ice nucleation activity even after it is detached from the cell wall, and is termed extracellular ice nucleator (ECIN). In this paper, surface morphology of ECINs was investigated using atomic force microscopy (AFM) in tapping mode. ECINs were immobilized onto polyelectrolyte multilayers using the layer by layer deposition method. Effect of layer build-up, method of ECIN production, and polyelectrolytes used for multilayer fabrication were investigated. Globular and rod-like structures were observed on the AFM images of the nano-thin ECIN layers. Activity of ECINs, tested in food solutions in earlier studies, was retained when applied as a nano-thin layer onto a silicon wafer surface. Protein aggregate sizes decreased when higher centrifugation speeds were applied, and ice nucleation activity also decreased. Nucleation occurred faster and at higher temperatures when substrates were immersed in solutions of higher ECIN concentration, whereas number of bilayers formed did not have a significant effect. Higher concentration ECIN dipping solutions also led to the formation of thicker and denser ECIN layers as observed via AFM imaging. PRACTICAL APPLICATION: This study demonstrates the properties of nano-thin ECIN layers, which can crystallize pure water into ice at higher temperatures and in shorter time. Accelerating ice nucleation can potentially be utilized to freeze liquids in shorter time hence using less energy, or improve frozen foods' stability against the risk of cold chain breakage.