Ultrastable PEGylated Calixarene-Coated Gold Nanoparticles with a Tunable Bioconjugation Density for Biosensing Applications

Many in vivo and in vitro applications using gold nanoparticles (AuNPs) require (i) their PEGylation, as it increases their stability and prevents nonspecific protein adsorption, and (ii) their conjugation to biomolecules, that provides them with specific recognition properties. Currently, the functionalization of AuNPs is based on thiol chemistry that suffers from two major drawbacks: (i) the Au-S bond is labile and confers limited chemical robustness to the organic layer, and (ii) control over the bioconjugation density is highly challenging. We report here a novel functionalization strategy based on calix[4]arene-tetradiazonium platforms for the coating of AuNPs with a robust PEG layer and their controlled bioconjugation. AuNPs were first modified with a functional calix[4]arene-diazonium salt bearing three PEG chains ended by a methoxy group and one by a carboxyl group. The resulting particles showed excellent chemical and colloidal stabilities, compared to similar systems obtained via a classical thiol chemistry, and could even be dispersed in human serum without degrading or aggregating. In addition to that, the carboxyl groups protruding from the PEG layer allowed their conjugation via amide bond formation with amine-containing biomolecules such as peptides. The control of the bioconjugation was obtained by grafting mixed layers of functional and nonfunctional PEGylated calix[4]arenes, that allowed varying the number of functional groups carried by the AuNPs and subsequently their bioconjugation capacity while preserving their dense protective PEG shell. Finally, we used these nanomaterials, modified with peptide aptamers, for the in vitro biosensing of a cancer biomarker, Mdm2.