This work describes the use of fluorescence correlation spectroscopy (FCS) and a novel amyloid-binding fluorescent probe, ARCAM >1, to monitor the aggregation of the Alzheimer's disease-associated amyloid β-peptide (Aβ). ARCAM >1 exhibits a large increase in fluorescence emission upon binding to Aβ assemblies, making it an excellent candidate for probe enhancement FCS (PE-FCS). ARCAM >1 binding does not change Aβ aggregation kinetics. It also exhibits greater dynamic range as a probe in reporting aggregate size by FCS in Aβ, when compared to thioflavin T (ThT) or an Aβ peptide modified with a fluorophore. Using fluorescent burst analysis (via PE-FCS) to follow aggregation of Aβ, we detected soluble aggregates at significantly earlier time points compared to typical bulk fluorescence measurements. Autocorrelation analysis revealed the size of these early Aβ assemblies. These results indicate that PE-FCS/ARCAM >1 based assays can detect and provide size characterization of small Aβ aggregation intermediates during the assembly process, which could enable monitoring and study of such aggregates that transiently accumulate in biofluids of patients with Alzheimer's and other neurodegenerative diseases.
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