Report
Teaser, summary, work performed and final results
Periodic Reporting for period 1 - ELSWIFLUOPRO (Electrochemically Switchable Fluorescent Probes for Biological Applications)
Teaser
The visualization of the cell-penetrating peptide translocation is of high importance. It can be achieved with fluorescence microscopy, which has become an essential tool for biological research. The bonding of a CPP to a small organic dye allows determination of the CPP...
Summary
The visualization of the cell-penetrating peptide translocation is of high importance. It can be achieved with fluorescence microscopy, which has become an essential tool for biological research. The bonding of a CPP to a small organic dye allows determination of the CPP localization and uptake. A wide range of fluorescent markers can be used for this purpose including rhodamine and fluorescein derivatives, BODIPY, or cyanine dyes. Nevertheless, the fluorescence labelling of CPPs with current fluorophores is connected with several drawbacks. The CPP uptake does not always correlate with bioavailability; therefore, the interpretation of the translocation process can be misleading. Moreover, CPPs are known to bind to the external face of the cell membrane and remain associated with cells even after repeated washings. The analysis can thereby give false positive results, since fluorescence cannot easily discriminate between internalized and surface-bound peptides.
To reduce signals from externally bound peptides, a protocol based on CPPs functionalized with nitrobenzoxadiazole (NBD) was developed. The outer NBD can be reduced by dithionite which results in its fluorescence quenching. The residual fluorescence of the sample, after the dithionite treatment, should therefore correspond to the intracellular species. However, dithionite must be used in excess, because it is air sensitive and undergoes rapid disproportionation in aqueous solution. Moreover, dithionite is not absolutely impermeant and may cross membranes, which leads to the extinction of fluorescence emitted by internalized peptides. In addition, once “switched offâ€, the NBD probe cannot be “switched on†due to the irreversibility of the reduction process. Therefore, search for a more reliable CPP-fluorophore system which would exhibite reversible on/off switching properties is very desirable.
The aim of this project is the development of novel versatile electrochemically switchable fluorescent probes for the detection of CPP translocation which is crucial within the context of the monitoring of drug delivery through cell membranes and, more generally, in biological research. The general idea is to couple a bright fluorophore with an electro-controlled fluorescent quencher and subsequently with a cell-penetrating peptide. So prepared CPP is expected to penetrate to the cell interior as well as bind to the external face of the cell membrane. The application of an adequate value of electric potential should result in the activation of the redox sensor and fluorescence quenching of photoactivated fluorophore through the photoinduced electron transfer (PET). As the electron transfer cannot occur through the membrane, the external probes would be switched off and fluorescence only of internalized CPPs could be study with high accuracy. The use of the redox sensor which could be reduced/oxidized reversibly would allow reversible on/off switching of the fluorescent probe.
Work performed
\"The results will be published in high impact factor international scientific journals:
1. ČÞková, M.; Cattiaux, L.; Mallet, J.-M.; Labbé, E.; Buriez, O. „Switching Fluorescence Emission in Rhodamine Derivatives\"\" ChemElectroChem submitted.
2. ČÞková, M.; Cattiaux, L.; Mallet, J.-M.; Labbé, E.; Buriez, O. „Rhodamine-Ferrocene based switchable dyad in cell imaging\"\" Angew. Chem. Int. Ed. in preparation.
3. ČÞková, M.; Cattiaux, L.; Mallet, J.-M.; Labbé, E.; Buriez, O. „Redox-controlled fluorescence modulation in NBD-Ferrocene derivative\"\" Electrochimica Acta in preparation.
The results were also presented in international conferences in order to increase the impact of the work:
1. RACI National Centenary Conference, Melbourne, Australia, 23-28 July, 2017.
2. 231st ECS Meeting, New Orleans, Louisiana, USA, 28 May - 1 June, 2017.
3. PRIME 2016, Honolulu, Hawaii, 2-7 October, 2016.
4. 67th Annual Meeting of the International Society of Electrochemistry, The Hague, Netherlands, 21-26 August, 2016.
Rhodamine derivatives
The photophysical and electrochemical properties of various rhodamine 101 derivatives have been investigated. The commercially available Rh101, used as a reference model, was first examined. Next, Rh101 was modified with an alkyne moiety. Subsequently, it was used for the synthesis of the triazole derivative. All three rhodamine derivatives were investigated by UV-Vis/fluorescence spectroscopy, cyclic voltammetry and UV-Vis/fluorescence spectroelectrochemistry. Fluorescence switching of Rh101 was achieved electrochemically. Electrochemical fluorescence extinction was successfully achieved also with both newly prepared derivatives. Moreover, it was shown that fluorescence of triazole derivative could be recovered electrochemically. This opened new perspectives in the use of rhodamine 101 derivatives as electrofluorochromic probes where the reactive bright fluorescent alkyne derivative could be utilized as a versatile and convenient unit for click functionalization with various partners including biomolecules. TD-DFT calculations were performed on the alkyne derivative and its reduced form.
Rhodamine-ferrocene derivative
Fluorescence of a rhodamine derivative was quenched by the attachment of ferrocene functional group through the photoinduced electron transfer (PET). Possibility to deactivate photoinduced electron transfer electrochemically leading to fluorescence switch on was examinated. First, fundamental optical and electrochemical properties of rhodamine- ferrocene derivative were studied by UV-Vis/fluorescence spectroscopy and cyclic voltammetry. Subsequently, fluorescence spectroelectrochemical experiments proved that PET in rhodamine-ferrocene compound can be electrochemically deactivated and fluorescence can be recovered.
NBD derivatives
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Final results
This multidisciplinary project is devoted to the development of versatile fluorescent probes which fluorescent properties can be activated and deactivated electrochemically. Such switchable fluorescent probes are of high interest within the context of the monitoring of drug delivery through cell-membranes and, more generally, in biological research. The work program relies on the combination of various scientific aspects such as (i) theoretical investigations, (ii) molecular design and synthesis, (iii) molecular electrochemistry, (iv) fluorescence, and will be ultimately applied to monitor the translocation of cell-penetrating peptides across artificial membranes. A certain number of important scientific breakthroughs can be anticipated as outcomes of this project such as (a) development of a synthetic chemistry targeted towards the preparation of original fluorescent probes bearing an electro-controlled fluorescent quencher, and (b) advances in analytical techniques related to molecular electrochemistry, fluorescence spectroscopy, and confocal microscopy.
Website & more info
More info: https://martinacizkova.wixsite.com/elswifluopro.