Controlled Temporal Release of Serum Albumin Immobilized on Gold Nanoparticles

Abstract

Proteins adsorbed to gold nanoparticles (AuNPs) form bioconjugates and are critical to many emerging technologies for drug delivery, diagnostics, therapies, and other biomedical applications. A thorough understanding of the interaction between the immobilized protein and AuNP is essential for the bioconjugate to perform as designed. Here, we explore a correlation between the number of solvent-accessible thiol groups on a protein and the protein desorption rate from the AuNP surface in the presence of a competing protein. The chemical modification of human serum albumin (HSA) was carried out to install additional free thiols using Traut's reagent and create a library of HSA analogues by tailoring the molar excess of the Traut's reagent. We pre-adsorbed HSA variants onto the AuNP surface, and the resulting bioconjugates were then exposed to IgG antibody, and protein exchange was monitored as a function of time. We found that the rate of HSA displacement from the AuNP correlated with the experimentally measured number of accessible free thiol groups. Additionally, bioconjugates were synthesized using thiolated analogues of bovine serum albumin (BSA) and suspended in serum as a model for a complex sample matrix. Similarly, desorption rates with serum proteins were modulated with solvent-accessible thiols on the immobilized protein. These results further highlight the key role of Au−S bonds in the formation of protein−AuNP conjugates and provide a pathway to systematically control the number of free thiols on a protein, enabling the controlled release of protein from the surface of AuNP.

Immobilization of Thiol-Modified Horseradish Peroxidase on Gold Nanoparticles Enhances Enzyme Stability and Prevents Proteolytic Digestion

“…39 Moreover, our group has previously reported that higher temperatures promote an increased denaturation rate of immobilized protein on AuNPs to establish a more robust interaction. 53 Proteolytic Degradation of Free and Immobilized Enzyme. The ability of an enzyme to resist protease digestion is critical to the realization of novel enzyme-based biotechnologies.…”

“…These cysteines are relatively solvent inaccessible; thus, it is unlikely that they interact with the AuNP unless the protein denatures. The solvent-accessible lysine residues on HRP can be exploited to install free thiols on HRP using amine-reactive chemistry to synthesize a thiolated HRP analog (THRP). ,,, Of the six lysine residues in HRP, it was previously established that three have side chains that are readily reactive via the primary amine (Lys174, Lys232, and Lys241), while the other three lysine residues are unavailable for modification because they are not exposed (Lys84), or sterically hindered by ion pairing and glycans (Lys65 and Lys149) …”

“…This unfolding/rearranging process can be relatively slow; the denaturation process for BSA on AuNPs was observed over a few days . Moreover, our group has previously reported that higher temperatures promote an increased denaturation rate of immobilized protein on AuNPs to establish a more robust interaction …”

γ-Functional Iminiumthiolactones for the Single and Double Modification of Peptides

“…33 With this small alteration comes a large increase in water solubility and reactivity toward primary amines at a physiologically compatible pH range of pH = 7−9. 34 Since its invention in 1973, the Traut's reagent has been continuously used to modify biological molecules (e.g., proteins, enzymes, ribosomes) and anchor them to surfaces, 35,36 tags, 37,38 or other (bio)macromolecules. 39,40 Despite its popularity, Traut's reagent can only be purchased as an unsubstituted fivemembered heterocycle.…”

“… With this small alteration comes a large increase in water solubility and reactivity toward primary amines at a physiologically compatible pH range of pH = 7–9 . Since its invention in 1973, the Traut’s reagent has been continuously used to modify biological molecules (e.g., proteins, enzymes, ribosomes) and anchor them to surfaces, , tags, , or other (bio)­macromolecules. , Despite its popularity, Traut’s reagent can only be purchased as an unsubstituted five-membered heterocycle. ITL derivatives with substitutions on the heterocycle have only been reported twice by Carroll and co-workers. , In the respective publications, the authors investigated the impact of various alkyl and phenyl substitutions on the reactivity and stability of the ITL derivatives.…”