Towards rational design: Developing universal freezing routes for anchoring DNA onto gold nanoparticles

[Display omitted] •Rational design approaches based on freezing interfacial interaction mechanisms have been studied, and spherical nucleic acids have been constructed using non-thiolated DNA.•Electrostatic repulsion and DNA secondary conformation were identified as the key factors affecting the anchoring efficiency of thiolated or non-thiolated DNA.•Several practical freezing strategies were provided to facilitate anchoring DNA onto AuNPs.•The extreme conditions required for freeze anchoring using non-thiolated DNA were further illustrated. DNA-functionalized gold nanoparticles (AuNPs), also known as spherical nucleic acids, are widely used in the development of biosensors, resulting in anchoring DNA onto AuNPs being a crucial preparation step and a popular research topic. The latest freeze-anchoring method is a simple and time-saving alternative to traditional salt aging; however, its universal applicability remains limited. In this study, we explored the interfacial interaction between DNA and the AuNP surface and proposed various universal routes for promoting freezing anchoring. Among them, rational design has been considered as the core idea to overcome these limitations, particularly using non-thiolated DNA anchoring, which offers significant advantages such as being unmodified, cost-effective, and easily accessible. We emphasize the importance of sequence structure and preparation process optimization, which mainly considers differences in DNA conformation and electrostatic repulsion. Additionally, the prepared DNA-functionalized AuNPs exhibited complete biological hybridization capability, and the extreme limiting conditions for non-thiolated DNA freeze anchoring were clarified. In summary, this study enhances our understanding of the interfacial relationship between DNA and AuNPs in the freeze-anchoring process and can significantly advance the applications of DNA-functionalized AuNP-based biosensors.