Freire Rodríguez, SoniaRodríguez-Prieto, FlorRíos Rodríguez, María del CarmenPenedo Esteiro, Juan CarlosAl-Soufi, WajihNovo, Mercedes2018-07-182018-07-182015Freire, S. , Rodríguez‐Prieto, F. , Ríos Rodríguez, M. C., Penedo, J. C., Al‐Soufi, W. and Novo, M. (2015), Towards Ratiometric Sensing of Amyloid Fibrils In Vitro. Chem. Eur. J., 21: 3425-3434. doi:10.1002/chem.201406110http://hdl.handle.net/10347/17053This is the peer-reviewed version of the following article: Chemistry - A European Journal 2015, 21, 3425–3434, DOI: 10.1002/chem.201406110. The final form has been published at https://onlinelibrary.wiley.com/doi/abs/10.1002/chem.201406110. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsThe aggregation of amyloid‐β peptide and its accumulation in the human brain has an important role in the etiology of Alzheimer’s disease. Thioflavin T has been widely used as a fluorescent marker for these amyloid aggregates. Nevertheless, its complex photophysical behavior, with strong wavelength dependencies of all its fluorescence properties, requires searching for new fluorescent probes. The use of 2‐(2′‐hydroxyphenyl)imidazo[4,5‐b]pyridine (HPIP), which shows two emission bands and a rich excited‐state behavior due to the existence of excited‐state intramolecular processes of proton transfer and charge transfer, is proposed. These properties result in a high sensitivity of HPIP fluorescence to its microenvironment and cause a large differential fluorescence enhancement of the two bands upon binding to aggregates of the amyloid‐β peptide. Based on this behavior, a very sensitive ratiometric method is established for the detection and quantification of amyloid fibrils, which can be combined with the monitoring of fluorescence anisotropy. The binding selectivity of HPIP is discussed on the basis of the apparent binding equilibrium constants of this probe to amyloid‐β (1–42) fibrils and to the nonfibrillar protein bovine serum albumin. Finally, an exhaustive comparison between HPIP and thioflavin T is presented to discuss the sensitivity and specificity of these probes to amyloid aggregates and the significant advantages of the HPIP dye for quantitative determinationseng© 2015 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim. This article may be used for non-commercial purposes in accordance with Wiley-VCH Terms and Conditions for Self-ArchivingAmyloid‐beta peptidesCharge transferFluorescent probesPhotophysicsProton transferjournal article10.1002/chem.2014061101521-3765open access