Amyloids are aggregates of proteins that can form in the human body and have been linked to many diseases, including Alzheimer’s and Parkinson’s. The structure of these aggregates can be studied using conventional and 2D infrared (IR) spectroscopy, but this can result in overlapping spectra in samples of amyloids mixed with protein monomers and oligomers.

Giubertoni et al. developed a method for studying mixed samples using IR diffusion-ordered spectroscopy (IR-DOSY). This method differentiates particles of different size and isolates the spectra from amyloids.

“Our method relies on the fact that the diffusion coefficient of a molecule is determined by its size,” said author Giulia Giubertoni. “Monomers diffuse much faster than aggregates, which can be up to a thousand times larger.”

The authors enabled this diffusion by creating a concentration gradient inside an IR sample cell. The mixed sample initially occupied half the cell, and they measured its equilibration over time. Due to the different diffusion coefficients, they could couple molecular size to the IR signal decay rate. Using this method, they performed both conventional and 2D IR spectroscopy with amyloid-monomer mixtures and demonstrated the isolation of amyloid spectra.

The team plans to apply their method to clinically relevant proteins and explore interactions involved in amyloid formation.

“As a next step, we would like to apply this method to investigate the aggregation of more ‘challenging’ proteins like alpha-synuclein, which is involved in Parkinson’s,” said Giubertoni. “It would also be interesting to use multi-dimensional IR-DOSY to study the effect of potential inhibitors on amyloid formation.”

Source: “Multidimensional infrared diffusion-ordered spectroscopy in depletion mode distinguishes protein amyloids and monomers,” by Giulia Giubertoni, Federico Caporaletti, Rianne van Diest, and Sander Woutersen, Journal of Chemical Physics (2023). The article can be accessed at

This paper is part of the Celebrating 25 Years of Two-dimensional Infrared (2D IR) Spectroscopy Collection, learn more here.