An effective intensity-based reference is a cornerstone for quantitative nuclear magnetic resonance (NMR) studies, as the molecular concentration is encoded in its signal. In theory, NMR is well suited for the measurement of competitive protein adsorption onto nanoparticle (NP) surfaces, but current referencing systems are not optimized for multidimensional experiments. Presented herein is a simple and novel referencing system using 15 N tryptophan (Trp) as an external reference for 1 H− 15 N 2D NMR experiments. The referencing system is validated by the determination of the binding capacity of a single protein onto gold NPs. Then, the Trp reference is applied to protein mixtures, and signals from each protein are accurately quantified. All results are consistent with previous studies, but with substantially higher precision, indicating that the Trp reference can accurately calibrate the residue peak intensities and reduce systematic errors. Finally, the proposed Trp reference is used to kinetically monitor in situ and in real time the competitive adsorption of different proteins. As a challenging test case, we successfully apply our approach to a mixture of protein variants differing by only a single residue. Our results show that the binding of one protein will affect the binding of the other, leading to an altered NP corona composition. This work therefore highlights the importance of studying protein−NP interactions in protein mixtures in situ, and the referencing system developed here enables the quantification of binding kinetics and thermodynamics of multiple proteins using various 1 H− 15 N 2D NMR techniques.

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The aggregation of Amyloid-b (Ab) peptides are known to be influenced by multiple environmental factors. Metals are one factor known to influence the self-assembly of Ab peptides. Studies have shown that copper and zinc ions are associated with increasing Ab peptide aggregation and plaque formation as observed in brain slices obtained from Alzheimer's patients. The mechanism of how metal ion interactions with Ab peptides alters self-assembly and the aggregation state has not yet been fully characterized. However, numerous studies performed under a variety of different experimental conditions have demonstrated metal ion effects on the aggregation of Ab (40) and Ab (42). Additional studies performed under identical conditions should allow for better characterization of how copper and zinc uniquely alter the different Ab peptides aggregation. These studies may provide a greater understanding of metal-induced Ab aggregation and how metals alter the development of neurodegenerative diseases. Infrared spectroscopy was used to monitor how metals altered Ab (40) and Ab (42). Infrared spectra were obtained on both peptides in the presence and absence of metals. The infrared spectra suggest metal-induced structural changes of the Ab-peptides as compared to control Ab(40) and Ab(42) spectra.