Quantum Dot-Antifreeze Protein Hybrids for Enhanced Ice Recrystallization Inhibition

Antifreeze proteins (AFPs) suppress ice growth by binding specific crystallographic planes, and their activity can be enhanced through multivalent display, where multiple AFPs simultaneously engage ice surfaces via multimerized constructs or nanoparticle scaffolds. However, the structural features of these scaffolds-and how they govern the degree of enhancement-remain poorly understood. Here, we present a systematic investigation of the design rules that control AFP performance when displayed on nanoparticle (NP) surfaces. We varied NP structural parameters, including size, geometry, curvature, and surface ligands, as well as AFP-NP interaction modes such as conjugation strategy and valency. Spherical gold nanoparticles (AuNPs), spherical quantum dots (QDs), and 2D QD nanoplatelets were used as scaffolds, and AFPs were attached through three methods: direct physisorption, indirect orthogonal conjugation via nitrilotriacetic acid-Ni2+ coordination, and direct orthogonal conjugation exploiting Zn2+-histidine affinity on Zn-rich QDs. Direct Zn2+-histidine conjugation produced the strongest ice recrystallization inhibition (IRI), outperforming both physisorption and Ni2+-mediated attachment. Control experiments with bovine serum albumin and NPs alone confirmed that the enhanced activity arose specifically from QD-AFP conjugates. We found that QDs with higher curvature and high surface-volume ratio exhibited superior IRI due to increased surface accessibility and less hindrance for AFP binding, while 2D nanoplatelets outperformed 3D QDs of similar volume by providing larger ice-binding interfaces. Our results revealed that AFP conjugation on QDs modified with non-branched shorter ligands showed enhanced IRI activity. Furthermore, low AFP-to-QD ratio yielded conjugates that exceeded the IRI activity of free AFPs by 16%. The intrinsic fluorescence of QDs enabled in situ visualization of ice shaping, revealing bright crystal edges upon AFP binding. Together, these results establish key structural principles for multivalent AFP display on nanomaterials and demonstrate that engineered NP-AFP hybrids can surpass native AFP performance, offering new opportunities in biotechnology, cryobiology, and antifreeze applications.