Real-Time Monitoring of Alzheimer’s-Related Amyloid Aggregation via Probe Enhancement–Fluorescence Correlation Spectroscopy

Abstract

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.

Solvation-Guided Design of Fluorescent Probes for Discrimination of Amyloids

“…Next, we measured the binding and fluorescence properties of these compounds in the presence of fully aggregated Aβ(1–42) peptides in solution (Fig. 5 ) 31 – 33 , 52 . All probes 1–5 were found to bind to aggregated Aβ with affinities in the low micromolar to high nanomolar range (see Supplementary Figure 4a–d for binding curves).…”

The effects of fluorescent labels on Aβ42 aggregation detected by fluorescence correlation spectroscopy

“…The aggregation and neurotoxicity of Aβ oligomers, especially for Aβ 42 oligomers, are subjects of intense AD research. Several techniques can be used to analyze Aβ oligomers, including fluorescence correlation spectroscopy (FCS), fluorescence resonance energy transfer, magic angle spinning (MAS) NMR spectroscopy (MAS NMR), total internal reflection fluorescence microscopy, SDS polyacrylamide gel electrophoresis (SDS‐PAGE), size‐exclusion chromatography, mass spectrometry (MS), dynamic light scattering, atomic force microscopy (AFM), and Thioflavin T spectroscopic assay …”

Identification of Patients with Preeclampsia by Measuring Fluorescence of an Amyloid-Binding Aryl Cyano Amide in Human Urine Samples

“…oligomers and fibrils). 19 Together, this strong sensitivity to PE supports that this probe has additional advantages as a screening tool for first-pass analyses of urine samples to identify PE.…”

“…We previously reported the structure and fluorescence properties of an amyloid-targeting aryl cyano amide molecular probe, ARCAM-1 (Figure 1A). 19 This dye, and its related family of fluorophores, exhibits a strong enhancement in fluorescence intensity and a hypsochromic shift (blueshift) of fluorescence emission in the presence of Aβ aggregates in solution and in tissue. 20–23 We also showed that this probe binds to amyloids comprised of different proteins, suggesting that its binding properties are associated with its capability to recognize common secondary structural characteristics common to all amyloids (e.g., high β-sheet content).…”