Published: June 30, 2011 r2011 American Chemical Society 11121 dx.doi.org/10.1021/ja204656r | J. Am. Chem. Soc. 2011, 133, 11121–11123 COMMUNICATION pubs.acs.org/JACS Sensing Amyloid-β Aggregation Using Luminescent Dipyridophenazine Ruthenium(II) Complexes Nathan P. Cook, † Veronica Torres, † Disha Jain, † and Angel A. Martí* ,†,‡ † Department of Chemistry and ‡ Department of Bioengineering, Rice University, 6100 South Main Street, Houston, Texas 77005, United States b S Supporting Information ABSTRACT: The aggregation of amyloid-β (Aβ) peptides has been associated with the onset of Alzheimer’s disease. Here, we report the use of a luminescent dipyridophenazine ruthenium(II) complex to monitor Aβ fibrillization. This complex is not photoluminescent in aqueous solution nor in the presence of monomeric Aβ, but it presents a strong photoluminescence in the presence of Aβ fibril aggregates. One of the advantages of this metal complex is its large Stokes shift (180 nm). Furthermore, the long-lived photo- luminescence lifetime of this ruthenium complex allows its use for the detection of fibrillar proteins in the presence of short-lived fluorescent backgrounds, using time-gating tech- nology. We will present evidence of the advantages of dipyridophenazine ruthenium(II) complexes for monitor- ing protein fibrillization in highly fluorescent media. A myloid-β (Aβ) is an extracellular peptide fragment, thought to play an important role in the pathology of Alzheimer’s disease (AD). AD is rapidly becoming a leading cause of death in the United States; an estimated 1116 million people are expected to be affected by 2050. 1 The amyloid cascade hypoth- esis suggests that the transition of monomeric Aβ to fibrillized- Aβ plays an important role in the pathology of AD, but the exact nature of Aβ toxicity is controversial. 2 Much work has been done in characterizing the transition of monomer to fibril, and perhaps the mostly widely used monitoring tool is thioflavin T (ThT). 3 ThT is a benzothiazole dye (Figure 1) that has minimal fluo- rescence (excitation 440 nm/emission 480 nm) in aqueous solu- tion or in the presence of monomeric Aβ; however, its fluores- cence increases by several orders of magnitude in the presence of fibrils. ThT being an amyloid binding dye, its derivatives have found important applications as potential in vivo diagnostic agents. 4 Dipyridophenazine ruthenium(II) complexes have been used in a wide variety of applications including DNA detection, 5,6 cell imaging, 7 DNA cleavage, 8 and photoinduced electron-transfer reactions, 9 among others. Recently, we reported that some of these complexes have the ability to disperse single-walled carbon nanotubes in aqueous solutions. 10 In this work, we report the light-switching properties of [Ru(bpy) 2 (dppz)] 2+ (bpy = 2,2 0 - bipyridine; dppz = dipyrido[3,2-a:2 0 ,3 0 -c]phenazine) when in contact with fibrillar Aβ. [Ru(bpy) 2 (dppz)] 2+ displays large Stokes shifts (180 nm) and long photoluminescence (PL) life- times, making it an ideal probe in highly fluorescent environments, both in vitro and ex vivo. The PL properties of dipyridophenazine Ru(II) and Ru(III) complexes have been characterized with some proteins, such as bovine serum albumin, 11,12 R-chymo- trypsin, 13 and certain transcription factors, 14 but to the best of our knowledge, [Ru(bpy) 2 (dppz)] 2+ light-switching properties have never been used to study peptide aggregation in real time. The PL response of [Ru(bpy) 2 (dppz)] 2+ upon protein ag- gregation is shown in Figure 1a. A typical sigmoidal behavior is observed for the PL of [Ru(bpy) 2 (dppz)] 2+ , in agreement with the fluorescence behavior of ThT. Generally, a lag phase is seen where Aβ monomers and soluble oligomers are present (throughout this paper we use the term “Aβ monomers” when referring to a freshly prepared non-aggregated Aβ solution which would contain a high concentration of monomeric Aβ but also some Aβ oligomers), and PL is not observed. After a few hours, protein aggregation starts, with the formation of small fibril aggregates which act as seeds for the further assembly of Aβ monomers into aggregated fibrillar structures. This aggregation and fibril elongation phase is relatively fast and followed by a leveling off of the PL once most of the monomers have been assembled into fibrils. The PL intensity of [Ru(bpy) 2 (dppz)] 2+ increases up to 50-fold in the presence of Aβ fibrils. The forma- tion of fibrils is confirmed by TEM images obtained from aliquots taken from the assays after 7 h (Figure 1b,c). The fluorescent behavior of ThT is modulated by rotation between its benzothiazole moiety and dimethylaniline group. 15 In aqueous solution the rapid rotation between these two groups provides a non-radiative deactivation pathway rendering a Figure 1. (a) Aβ fibrillization assay with ThT (blue ]) and [Ru(bpy) 2 - (dppz)] 2+ (red b). Right: TEM images of Aβ fibrils after 7 h incubation in the presence of (b) ThT and (c) [Ru(bpy) 2 (dppz)] 2+ . Scale bar = 100 nm. Received: May 20, 2011