Realization of innovative fluorochromes is an essential requirement to push advances in bioimaging and diagnostics. Once endowed with desired optical properties, they have to be integrated into standard staining technologies for biological materials. There still exist different classes of dyes which show complementary qualities and drawbacks.
This project aims to develop a new family of fluorescent molecules for highly sensitive chemical and biological detection that merge the incomparable brightness of pi-conjugated polymers, in our case Violanthrone, to the narrow emission lines of lanthanide elements.
Transient Absorption measurements, performed in Femtoscopy Labs at Sapienza in Rome, will probe the main mechanisms that characterise the photophysics of such complexes, e.g. excimer formation and charge transfer. The study of ultrafast photoinduced dynamics and the determination of the characteristic timescale will allow to determine the best chemical functionalization to obtain the-state-of-the-art fluorescent dyes.
The full characterization of Violanthrone represents a fundamental step in order to integrate this polymer in a polyaromatic antenna able to be exploited as target with model features for biological imaging. The very last goal of this project is to assemble this supramolecular complexes within the internal cavity of a novel, engineered ferritin protein platform very recently developed at Department of Biochemistry (Prof. Boffi) [1,2]. As such, the optically active molecules will be confined within the protein cavity which also provide specific binding sites for lanthanide atoms thus conferring additional unique biocompatibility properties to the system.
In a broader perspective, the application of this project is deeply linked to the very recent development of a set of ferritin protein mutants endowed with unique assembly-disassembly properties coupled to high affinity lanthanide binding properties. In particular two genetically engineered constructs have been obtained: 1) humanized archaeal ferritin (HumAFt) and 2) Lanthanide binding ferritin (LnHFt). Notable properties of both HumAFt and LnHFt will be merged into one or more construct endowed with convenient auto assembly properties together with specific lanthanide binding properties. The new construct will be able to host lanthanide ions within well-defined topological positions inside the ferritin cavity and pi-coniugated polymers will be encapsulated into such construct by Mg++ dependent protein assembly.
The resulting ferritin constructs will be able to host the polyaromatic antenna system within the 10 nm cavity in close contact with lanthanide ions directly coordinated to the protein through highly specific aminoacid binding loops. As such, two photophysical phenomena may be generated depending on the exciting source and on the nature of the antenna system: i) the conjugated aromatic polymer will be able to absorb light and transfer energy directly to the lanthanide atom for delayed emission in the near infrared, or ii) the cyanine dye will harvest infrared light and transfer two photons to the lanthanide atom for doubled energy emission in the uv-vis (upconversion).
In both cases, antenna systems with enhanced cross sections (broad absorption bands and high molar absorptivities), short lifetimes and high stability towards photobleaching will harvest photons and subsequently transfer them to lanthanide ions. Lanthanides, endowed with much longer lifetimes, will then emit radiation in much narrower lines. Confinement of 20-40 dye molecules and 92 lanthanide atoms within the 300 A 3 ferritin cavity is deemed to be a sufficiently tight packaging to allow efficient photon transfer, as shown in fig. 2
.
Clearly, once the ferritin construct will be realized, ultrafast spectroscopy will have a pivotal role to unveil the charge transfer dynamics. In particular, it is able to estimate the transfer efficiency and the characteristic timescale.
As such, the water soluble, highly biocompatible ferritin construct may be used for direct targeting to CD71 cell receptor or readily modified with easy methods (either genetic or chemical) to confer diverse specificity to the desired receptor. The project has a high technology transfer content, due to the enormous commercial interest in research and diagnostics for such bright fluorescent labels. Several patented technologies are gaining progressive market shares in recent years and a novel technological solutions related to these fields may lead to competitive fluorochromes.
[1] V. de Turris et al, Nanoscale. 2017 Jan 5;9(2):647-655.
[2] L. Calisti et al., Biochim Biophys Acta. 2017 Feb;1861(2):450-456.