Examining Amyloid-β Structure During the Progression of Alzheimer's Disease using an Immuno-2DIR- Sensor Abstract Alzheimer's disease is estimated to affect 30 million people and is the most common cause of dementia. It is known that the disease is associated with plaques formed by Aβ aggregating into long β-sheet-rich fibrils. The disease progression is probably associated with the formation of Aβ oligomers that are toxic to neurons. However, obtaining structural information about these oligomers is exceedingly difficult, because they reside in very small concentrations in the body. The purpose of this proposal is to develop an assay that can monitor the structural distributions of Aβ extracted from serum and cerebrospinal fluid of healthy and Alzheimer's inflicted individuals. The proposal is made possible by a new breakthrough in the sensitivity of 2D IR spectroscopy, enabling data collection on a single monolayer of proteins. This new capability allows us to measure the 2D IR spectra of Aβ polypeptides that are extracted from blood using antibodies in a flow-through sample cell. Indeed, our approach is derived from very exciting ATR-FTIR reports that recently found that the amide I frequency of Aβ is correlated to the progression of the disease, which indicates that the distribution of structures of Aβ evolves as the disease progresses. The proposed 2D IR assay will be more sensitive and provide more structural insight than the original ATR-FTIR work. Aim 1 of this proposal is to develop the 2D IR version of the assay and use it to measure in vitro generated monomers, oilgomers, and fibrils of Aβ polypeptides. These experiments will give insight into the biophysical mechanism of Aβ aggregation, especially low population species that can play outsized roles in the nucleation of Aβ fibers, as well as generate a library of reference 2D IR spectra. Aim 2 will be to use the assay to extract Aβ from blood plasma and cerebral spinal fluid to monitor the structure of Aβ with the progression of the disease. Samples will be provided by the Wisconsin Alzheimer's Disease Research Center. The libraries from Aim 1 will help parse the structural distributions. This proposal is high risk because it utilizes a brand new technology, but also has the potential for high impact, since the structural insights that promise to be gained on in vivo Aβ would be difficult, if not impossible, to obtain any other way.