Optimization of the Gold Nanoparticle Colorimetric Assay for Screening and Quantifying Ice Recrystallization Inhibition Activity of Antifreeze Proteins

Antifreeze proteins or ice-binding proteins can be found in a variety of organisms, including fish, insects, and plants. These proteins are responsible for hindering ice crystal growth, a process known as ice recrystallization inhibition (IRI), allowing the organism to survive in subzero temperatures. Antifreeze proteins are an attractive area of research because of their potential applications in wide-ranging areas, such as food and crops preservation, industry, and cryopreservation. Since antifreeze proteins are not always suitable for these applications due to proteolytic degradation and/or loss of activity after immobilization, there is a growing demand for antifreeze protein mimics. The identification and development of these mimics is impeded by a suitable screening method to rapidly detect and identify novel agents with ice recrystallization inhibition activity. Here, we report an optimized gold nanoparticle colorimetric assay for the screening of IRI activity, with ligand-functionalized freeze-labile gold nanoparticles. A new method was developed for data processing and quantification of the IRI activity by making use of the Hill equation. A variety of different antifreeze proteins were selected to be subjected to the colorimetric assay, and each showed a strong dose-response correlation, showcasing the robustness and versatility of this assay.