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Stimulated emission from rhodamine 6G aggregates self-assembled on amyloid protein fibrils Piotr Hanczyc, Lech Sznitko, Chengmei Zhong, and Alan J. Heeger ACS Photonics, Just Accepted Manuscript • DOI: 10.1021/acsphotonics.5b00458 • Publication Date (Web): 11 Nov 2015 Downloaded from http://pubs.acs.org on November 14, 2015
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Stimulated Emission From Rhodamine 6G Aggregates Self-Assembled on amyloid Protein Fibrils Piotr Hanczyc1, Lech Sznitko2, Chengmei Zhong1, Alan J. Heeger1* 1. University of California, Santa Barbara, Center for Oligomers & Organic Solids, 2520A Physical Sciences Building North, Santa Barbara, United States. 2. Wroclaw University of Technology, Advanced Materials Engineering and Modelling Group, Faculty of Chemistry, 50-370, Wroclaw, Poland.
*correspondence to:
[email protected] Abstract Amyloid fibrils are excellent bioderived nanotemplates for controlling molecular and optical properties of small molecules such as organic dyes. Here we demonstrate that two representative fibril-forming proteins, lysozyme and insulin from the amyloids family can determine the optical signature of rhodamine 6G. Their structural variety leads to a unique molecular arrangement of dye aggregates on the biotemplate surface.
This significantly
influences the light amplification threshold as well as the stimulated emission profiles which show remarkable broadband wavelength tunability. We show in addition that amyloid fibrils can be potentially used in constructing broadband emission biolasers.
Keywords: Amyloid fibrils, lysozyme and insulin proteins, Stimulated emission, Random lasing, Laser dye, Rhodamine 6G, bio-laser, biomaterial, nano-templates, molecular crowding
Amyloid fibrils are a hallmark of neurodegenerative diseases1, 2. Since their discovery, research has focused on understanding their formation, progression and structural features3. Molecular studies indicate that amyloid fibrils are unique biopolymers with rich diversity where
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β-sheet structures are predominant 4. The studies revealed also their interesting material properties where rigidity of a single fibril of different protein source can range from highly flexible (90–320 MPa)5 to extremely stiff (2–14 GPa) 6. The diversity and self-assembling properties of amyloid fibrils makes them attractive nanomaterials for supramolecular complexation of small molecules. In this article we expand our previous research on amyloid-dye complexes7 to two types of amyloid forming proteins, insulin and lysozyme that were doped with Rhodamine 6G. Organic fluorophores are well-known for high affinity to amyloid fibrils
8-11.
However, the rhodamine
family of dyes is particularly interesting because of both medical and technological relevance. Its derivatives have been used as staining agents, for example monitoring the drug uptake at blood brain barrier in Alzheimer therapy12. On the other hand they are suitable and often fluorophores of choice for laser construction because of convenient photophysical features13. Here we show that Rhodamine 6G stimulated emission profiles are related to the intrinsic structure of the amyloid protein fibrils and their material properties. Rhodamine 6G interacting with fibrils enables extending classical spectroscopic characterization of amyloid-dye complexation using laser spectroscopy as was presented previously for DNA14-16 and silk fibroins 17, 18. Our experiments indicate that the biophotonics that was previously related only to DNA and
DNA complexes, can be expanded to a broader spectrum of biologically derived materials such as amyloid forming proteins. We show that not only processes between biomolecules and chromophores (e.g. intercalation, groove binding) may be utilized to tailor photonic properties of biomaterials but also, molecular crowding and interactions with second order structures of these materials are relevant to the optical signatures. Proteins are particularly interesting due to their ability of generating higher order species through self-assembly. Moreover, there are many possible interactions with different dyes as a result of electrostatic and/or Van der Waals interactions. These different supramolecular complexes may become crucial for designing future bio-based materials for photonics. Moreover they are bio-friendly, the production does not require oil or other non-renewable sources and often they can have a diagnostic capability. Here we show that stimulated emission spectra and random lasing provide information about chromophore-chromophore interactions within the binding sites in the supramolecular
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complexes. Fluorophore stimulated emission (SE) spectra significantly vary depending on the protein source. Therefore the SE spectra provide indirect information about the intrinsic structure of amyloid fibrils. Since these two types of amyloid forming proteins resemble the structure of the vast majority of disease related pathogenic specimens, stimulated emission and random lasing might provide additional information in biosample analysis e.g. recognition of various amyloids in cerebrospinal fluids (CSF). The structural polymorphism of amyloid fibrils affects the thin film morphology and thereby the mechanism of light amplification. Molecular crowding enhances the optical signals and shows the potential of amyloid fibrils in technological applications e.g. bioderived lasers where biotemplates intrinsic properties can determine the function of biodevices. The key finding of our work is the demonstration that the type of amyloid fibrils determines the size and shape of rhodamine 6G aggregates at bioderived nanotemplate surface, as monitored by recording the fluorophore stimulated emission spectra.
Materials and Methods Experimental section: Native monomers of lysozyme protein from chicken egg white and insulin protein from bovine pancreas were purchased from Sigma Aldrich and dissolved to 10 mg/ml in pH=2 (0.01M HCl) water and used as the stock solution concentration. The solutions were filtered through 0.2 μm filter and heated to 65 oC for 6 days and 24 h, respectively. After fibril formation the samples were centrifuged at 3000 rpm for 3 min in order to remove globular particulates. The stock solution of rhodamine 6G dye was prepared by dissolving 5 mg/ml of the laser dye in water. For all solution experiments the stocks of Rh6G and amyloids were diluted to 0.05 mg/ml and 0.1 mg/ml respectively. For thin film preparation, dye and amyloid fibrils stock solutions were mixed in 1:20 of dye to biotemplate volume ratio and left for 1 h. The solid state samples were prepared by casting on silica and left for drying for 24 h in ambient conditions. UV-Vis absorption spectra of amyloids-rhodamine complexes were recorded on a CARY-5000 spectrophotometer. Linear Dichroism: The LD is defined as the difference in absorbance of light linearly polarized parallel (A∥) and perpendicular (A⊥) to the macroscopic axis of orientation: 3
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(1)
−
The LD spectra of amyloid fibrils doped with rhodamine 6G were recorded using a Chirascan CD spectrophotometer equipped with quartz Couette cell for aligning the solute samples. In order to calculate the angle between biopolymer macroscopic axis and bound chromophore the reduced LD (LDr) is necessary. LDr is a product of an orientation factor 0
