Polymer-tethered glyconanoparticle colourimetric biosensors for lectin binding: structural and experimental parameters to ensure a robust output

Glycan-lectin interactions play essential roles in biology; as the site of attachment for pathogens, cell-cell communication, and as crucial players in the immune system. Identifying if a new glycan (natural or unnatural) binds a protein partner, or if a new protein (or mutant) binds a glycan remains a non-trivial problem, with few accessible or low-cost tools available. Micro-arrays allow for the interrogation of 100's of glycans but are not widely available in individual laboratories. Biophysical techniques such as isothermal titration calorimetry, surface plasmon resonance spectrometry, biolayer interferometry and nuclear magnetic resonance spectroscopy all provide detailed understanding of glycan binding but are relatively expensive. Glycosylated plasmonic nanoparticles based on gold cores with polymeric tethers have emerged as biosensors to detect glycan-protein binding, based on colourimetric (red to blue) outputs which can be easily interpreted by a simple UV-visible spectrometer or by eye. Despite the large number of reports there are no standard protocols for each system or recommended start points, to allow a new user to deploy this technology. Here we explore the key parameters of nanoparticle size, polymeric tether length and gold concentration to provide some guidelines for how polymer-tethered glycosylated gold nanoparticles can be used to probe a new glycan/protein interactions, with minimal optimisation barriers. This work aimed to remove the need to explore chemical and nanoparticle space and hence remove a barrier for other users when deploying this system. We show that the concentration of the gold core is crucial to balance strong responses versus false positives and recommend a gold core size and polymer tether length which balances sufficient colloidal stability and output. Whilst subtle differences between glycans/lectins will impact the outcomes, these parameters should enable a lab user to quickly evaluate binding using minimal quantities of the glycan and lectin, to select candidates for further study.