Spectroscopic Investigation of DCCH and FTSC as a potential pair for Förster Resonance Energy Transfer in different solvents

Abstract

Two molecules, 7-(diethylamino)coumarin-3-carbohydrazide (DCCH) and fluorescein-5-thiosemicarbazide (FTSC) were investigated in different solvents, under varying pH conditions regarding their spectroscopic properties for the usage as a Förster Resonance Energy Transfer (FRET) pair to study the molecular interaction between cellulosic surfaces. All the relevant spectroscopic properties to determine the Förster distance were measured and the performance as a FRET system was checked. From the results, it is clear that the environmental conditions need to be accurately controlled as both, but especially the FTSC dyes are sensitive to changes. For high enough concentrations positive FRET systems were observed in DMF, DMSO, H2O, THF and alkaline DMF. However due to the low quantum yield of the unmodified DCCH throughout the investigated parameter range and the strong environmental dependency of FTSC, both dyes are not preferable for being used in a FRET system for studying interaction between cellulosic surfaces.

Fig 1. Molar attenuation coefficient of DCCH and FTSC at different pH conditions and in various solvents.

a,b) Attenuation Coefficient of DCCH. The coefficients vary up to a factor of approx. 2.5 in the various solvents but do not show much change due to pH except for THF. The Toluol labeled sample represents the measurement of the reference molecule Coumarin 30 for the quantum yield (QY) measurements. c,d) Attenuation coefficient of FTSC. NaOH is the measurement of Fluorescein Sodium Salt needed as a reference for the QY of FTSC. c) One can see that the absorbance of FTSC is almost fully quenched in DMF, DMSO and THF in the visible range. In H₂O the performance is a little better. d) Going to alkaline conditions increases the attenuation coefficient in most solvents.

Fig 2. Chemical modification of the hydrazide group by acetylation with acetyl chloride.

The resulting hydrazine increases the QY of the Coumarin by a factor of 5.

Fig 3. Normalized excitation and emission spectra of DCCH and FTSC in different solvents and pH values.

In a) and b) one can see the spectra in water under neutral and alkaline conditions. One important feature in c) and d) is that in the neutral solution the FTSC spectra shift strongly (130–150 nm) into the blue compared to the H₂O spectra. In the alkaline solution the spectra shift back to the red and exhibit many side excitations.[22].

Fig 4. Normalized excitation and emission spectra of DCCH and FTSC in different solvents and pH values.

In a) and b) one can see the spectra in DMF. Here the FTSC EX/EM shifts to 340/410 nm in the neutral case and shifts back to higher wavelengths (520/540 nm) in alkaline conditions where it additionally exhibits a second emission at 410 nm. The DCCH main excitation becomes narrower and a second excitation at 370 nm appears in the neutral state. Going to alkaline conditions shifts the DCCH spectra into the blue. c) and d) show the spectra in DMSO. The spectra for FTSC look quite similar as in DMF with the exception of the missing additional emission at 410 nm in the alkaline state. The DCCH spectra for the alkaline conditions are similar while in the neutral state the excitation spectrum shifts a few nm into the blue and the spectra become wider.

Fig 5. Fluorescence measurements with a concentration of 0.1 mM.

When the dyes are mixed together the FTSC fluorescence is quenched and the DCCH fluorescence is enhanced which is a clear sign of FRET.

Fig 6. FRET measurements using a capillary and thus going to higher concentrations.

Due to the higher concentration a FRET signal is also detected under other conditions. (Conc. H₂O = 0.15 mM, conc. Other = 1 mM).