Amyloid diagnostics: Probing protein aggregation and conformation with ultrasensitive fluorescence detection

Abstract

While dozens of human ailments are now identified as "protein aggregation diseases", aggregation by itself does not seem to be a clear determinant of the toxicity. The structural transformation that accompanies the initial steps of aggregation may be an even more important aspect controlling the biological effects of these protein particles. For this, the key is to develop appropriate fluorescent biomarkers which can probe both aggregation and conformation at low physiological concentrations. Using Alzheimer's amyloid beta (Aβ) as a model system, we have developed probes suitable for the application of Fluorescence Correlation Spectroscopy (FCS, which reports aggregation) and Förster Resonance Energy Transfer (FRET, which reports conformational changes) techniques. To diagnose these changes in the cerebrospinal fluid of Alzheimer's patients, we are now designing better single molecule detection devices. Here we report a confocal device with a 4π collection geometry, which detects more than 0.5 million photons per second from a single rhodamine B molecule in aqueous solution, which to our knowledge is the highest sensitivity achieved so far with such devices. This allows us to perform quick and sensitive antibunching measurements which report the aggregate mass and fluorophore lifetime of Aβ oligomers.