Revival, enhancement and tuning of fluorescence from Coumarin 6: combination of host–guest chemistry, viscosity and collisional quenching

Abstract

Fluorescence from Coumarin 6 (C6), a laser dye, decreases considerably due to microcrystal formation in aqueous environments. The fluorescence yield can be effectively enhanced by applying β-cyclodextrin that revives the molecular entity of C6 through host–guest chemistry. C6-β-CD capsules form nanocubes in solution with surface projected hydroxyl groups. Increase in solvent viscosity brings the nanocubes closer following agglomeration, while keeping the molecular entity of C6 intact. This enhances the fluorescence from C6 encapsulated in β-CD nanocubes by 40%. Moreover, this emission can be tuned quantitatively by applying nanoparticles (silver in the present case) at each environmental viscosity level.

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Coumarin 6 (C6) is a well-known laser dye that is widely used in fluorescence spectroscopy.^1–4 On one hand, C6 has extensive optical applications and, on the other, this dye is also used in pharmaceutical purposes.^5,6 C6 is found to form microcrystals, even at very low concentration (∼1 μM), in aqueous environments on incubation at room temperature within a short period of time.⁷ The microcrystal formation, however, is unwanted from the application point of view of C6. The phenomenon profusely reduces fluorescence yield of C6 due to parallel stacking of the molecules and loss of energy through non-radiative channels. In a previous work, we have shown that the C6 aggregates can be effectively broken using cyclodextrin (CD) molecules of β- and γ-varieties, i.e., smaller and bigger cavity size.⁷ It was explained that due to encapsulation of C6 inside the relatively hydrophobic CD cavities, the molecule regains its singular entity and hence the fluorescence yield increases remarkably. Enhancement in C6 fluorescence was more for β-CD encapsulation as the cavity allows only one molecule to enter. Microscopy suggests that C6 included β-CDs form nanocubes due to self-aggregation through formation of hydrogen bonds using the projected hydroxyl groups on the rims of β-CD (Fig. 1).⁷ The nanocubes can be well-identified in the aqueous environment that leads to disintegration of the C6 microcrystals and enhance the fluorescence yield of C6 through revival of their molecular entity.

Fig. 1 Host–guest encapsulation schematic and scanning electron micrograph of C6 in β-CD.

C6 forms 1 : 2 guest–host complex at low concentrations of β-CD that eventually undergo hydrogen bonding at the outer rims of the CD to provide the nanocubes at higher CD concentration.^8–10 The 1 : 2 stoichiometry for C6-β-CD is confirmed by a double reciprocal Benesi–Hildebrand plot that shows a linear fit according to the equation (Fig. 2):¹¹

where, I₀ and I_m are the fluorescence intensities at zero and maximum concentration of β-CD, I denotes the fluorescence intensities at different concentrations of β-CD, [β-CD] is the total CD concentration, and K is the binding constant. The binding constant, K, comes out to be 46 × 10³ M that shows strong guest–host binding.

Fig. 2 Benesi–Hildebrand double reciprocal plot to show 1 : 2 stoichiometry between C6 and β-CD. The CD concentration has been varied from 0 to 1.5 mM and concentration of C6 is 1 μM.

The C6-β-CD nanocubes fluoresce at 506 nm and do not show any trace of microcrystalline aggregation of C6.⁷ Revival of the faded fluorescence of C6 could bring back its applicability in the optical world as also pharmaceutical scenario. Due to the fluorescing character of C6 as a result of intramolecular charge transfer,¹² this compound can be used as an efficient fluorescent marker.^5,13 Hence, in the present study we have endeavored to apply variable viscosity to the aqueous environment of the C6-β-CD nanocubes with an aim to further enhancement of their fluorescence yield. While looking at the changes in fluorescence of the nanocubes at different solvent viscosity, we have used biocompatible silver nanoparticles (AgNPs) to tune the emission quantitatively so that the property can be used vividly.

It is known that viscosity of water–glycerol mixture increases exponentially after reaching 50% by volume.¹⁴ Since increase in viscosity of the medium will create motional restriction to the molecular entities, hence the fluorescence yield is supposed to increase. The principal aim of the present study is to convert C6 as a better laser dye with tunability in fluorescence intensity as and when required.

Steady state measurement of fluorescence from C6-β-CD nanocubes shows a band maximum at 506 nm. Comparison with self-quenched emission from C6 microcrystals in water indicates elimination of C6 aggregation due to host–guest interaction (Fig. 3). Once the emission with much higher yield is achieved, it is found to be stable for an extensive period of time. Progressive red shift in the emission maximum of C6-β-CD is observed until the glycerol concentration reached 50% by volume. The shift stopped after 50% glycerol concentration followed by a concomitant increase in fluorescence yield as shown in Fig. 4. The initial decrease in fluorescence is presumably due to collision among the nanocubes that ceases on reaching a particular viscosity and find longer residence time to form mutual hydrogen bonds. While the initial red shift indicates gradual stabilization of the system due to increased motional restriction, the stoppage in emission shift after 50% glycerol indicates initiation of formation of hydrogen bonds among the nanocubes leading to association. Fluorophores lose their fluorescence yield through non-radiative dissipation of the absorbed energy on having more degrees of freedom in solution. Decreased motional restriction will reduce such loss in energy and hence the excited singlet state gets more stabilized which leads to the red shift in fluorescence spectrum.

Fig. 3 Fluorescence spectrum of C6 in water with and without β-CD encapsulation. The inset gives a magnified view of emission from the C6 microcrystals. The black arrow indicates formation of a band at 560 nm due to aggregation.

Fig. 4 Fluorescence spectra of C6-β-CD at various glycerol concentrations. The legend indicates percentage of glycerol in aqueous mixture.

The schematic in Fig. 1 shows the host–guest pattern of C6-β-CD composite. Presence of hydroxyl groups on the rims of β-CD makes them open toward thriving through further hydrogen bonding interactions as the conditions may be. Increase in solution viscosity by adding glycerol to water is shown in Fig. 5(a) and the change in relative fluorescence from the C6-β-CD nanocubes is shown in Fig. 5(b). It can be easily observed that small change in solvent viscosity till 50% glycerol composition, results into lowering of fluorescence yield of the C6-β-CD nanocubes. However, the steady state fluorescence anisotropy of C6 shows small enhancement during this viscosity change indicating inception of motional restriction as shown in Fig. 5(c). After 50% glycerol composition the anisotropy increases sharply to reach 0.33 at 100% glycerol composition providing evidence for the suprastructure formation.

Fig. 5 (a) Change in viscosity of water at different concentrations of glycerol, (b) relative change in fluorescence yield of C6-β-CD nanocubes, and (c) change in fluorescence anisotropy due to alteration in solution viscosity.

The initial quenching in fluorescence may result due to collision among the nanocubes leading to dissipation of energy. Rapid enhancement in solvent viscosity above 50% glycerol composition shows remarkable enhancement in fluorescence intensity of the C6-β-CD nanocubes. This is achieved presumably due to formation of suprastructures because of proximity of the slow-moving nanocubes in solution and progressive union due to hydrogen bond formation between the projected hydroxyl groups of the β-CDs in the nanocubes. The suprastructure formation will supposedly lead to reduction in non-radiative dissipation of energy resulting to progressive enhancement in fluorescence yield. Fig. 6 shows the different phases of association of the C6-β-CD nanocubes at various glycerol percentages in aqueous mixture. Formation of suprastructure due to hydrogen bonding between the nanocubes is quite clearly observed at 100% glycerol.

Fig. 6 SEM images of arrangement of C6-β-CD nanocubes at (a) 50%, (b) 70%, and (c) 100% glycerol concentration (v/v). A magnified view of the suprastructure formation is provided at 100% glycerol concentration in (d).

The results so far could show that C6 emission can be increased by about 40% above what was obtained by encapsulating it inside β-CD cavity thus preserving its molecular entity to prohibit fluorescence quenching due to microcrystal formation. We further looked into possibility of tuning the fluorescence yield at each glycerol concentration in aqueous mixture by externally adding AgNPs of about 100 nm diameter. Fig. 7 shows that tuning of C6-β-CD fluorescence can be effectively done by the added nanoparticles through collisional quenching. Increase in viscosity reduces the quenching constant due to viscous environment.

Fig. 7 Quantitative quenching of C6-β-CD fluorescence by AgNPs at different concentrations of glycerol shown in percentage in the legend. The quencher concentration was varied from 0 to 0.4 mM.

In summary, we have demonstrated that the fluorescence property of C6, which is used widely as a laser dye, can be revived through formation of β-CD nanocubes.⁷ The fluorescence yield can be further increased by 40% by regulating the solvent viscosity keeping the molecular entity of C6 intact. The emission can be further quantitatively fine-tuned by externally adding regulated concentration of AgNPs. Thus, the resulting device can be effectively used as an efficient and stable laser dye whose fluorescence yield can be tuned as desired. This device can also be applied in biosystems as markers under various viscosities.

Acknowledgements

PP acknowledges financial support from Science and Engineering Research Board, Department of Science and Technology, Government of India through project number EMR/2015/000950. SM and RB thank University Grants Commission and IISER Kolkata for fellowships.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and additional data. See DOI: 10.1039/c6ra20884c

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