4.3 Results and Discussion
4.3.3 Fluorescence Spectroscopy Experiments
The fluorescence spectra in dichloromethane (λexc 330 nm, Figure 4.13) of compound XVII validated our new design, in fact an excimer emission band can be observed centered at about 480 nm.
Figure 4.13 Fluorescence spectrum of XVII in dichloromethane (c = 2 x 10-6 M).
The sensor for illicit drugs sensing detection must operate at the solid-water interface. The multi-aromatic cavity of the tetraphosphonate receptor and the two pyrene moieties attached at the top of the molecule make our receptor extremely hydrophobic. In water XVII is thus not soluble, forming a dispersion of aggregates with a diameter of ca. 100 nm. To overcome this issue, we used
77 the strategy to load the cavitand molecules in silica nanoparticles having an outer PEG shell (Pluronic-Silica NanoParticles -PluS NPs), a strategy that was conveniently used with other chemical sensors.44 In this case, we modified the synthesis of the NPs using a modified silica precursor (1,2-bis(triethoxysilyl)ethane, Figure 4.14b) in place of the commonly used tetraethoxysilane (TEOS, Figure 4.14a), in order to increase the loading ability of the NPs structure (L. Petrizza, unpublished results).
Figure 4.14 a) TEOS; b) 1,2-Bis(triethoxysilyl)ethane.
In addition, the use of a confined environment was conceived also to increase the formation of an intramolecular excimer as observed in other system like micelles,45 bilayers,46 zeolites,47 sol-gel materials and glasses.48
Figure 4.15 Fluorescence spectrum (λexc 330 nm) of XVII + nanoparticles in H2O (c= 2 x 10-6 M).
Interestingly, dynamic light scattering measurements of equimolar solutions of XVII and PluS NPs show only the typical, very narrow peak of the
nanoparticles, indicating the absence in these conditions of self-aggregation of XVII due to their loading into the structure of the nanoparticles. The fluorescence spectrum of the cavitand loaded nanoparticles in water (Figure 4.15) clearly shows, as expected, a large contribution from the excimeric emission, indicating that, by large, the major part of the excited states of pyrene decay to their ground state through the formation of excimers. NPs thus play here a major role in providing the best starting condition for fluorescence sensing: aggregation of the chemosensor is satisfactorily avoided, and we obtain stable and intense excimer emission.
We chose four different guests to test our chemosensor in aqueous solution:
amphetamine, MDMA, cocaine and 3-fluoro-methamphetamine (FMA, a designer drug) all of them hydrochloride salts. Such guests were selected as representatives of the ATS and natural illicit drugs classes. FMA, in particular, is a representative of a so called “designer drugs”. We then selected several control compounds in order to rule out possible interferences in the sensing procedures: glucose since it is commonly used as the excipient in street samples, glycine as an amine which is not hosted by the cavitand, and, sarcosine as control because, although it presents affinity towards the receptor, it does not have a bulky unit such as the illicit drugs, therefore it should not trigger any response.
Figure 4.16 Fluorescence intensity in the excimer band (λexc = 330 nm; λem = 475 nm) of XVII + nanoparticles in H2O (c = 2 x 10- 6 M) upon addition of increasing amounts of
79 Interestingly, we observed large spectral changes upon addition of MDMA, amphetamine, cocaine, and FMA hydrochloride salts while control compounds sarcosine, glucose and glycine did not substantially affect the emission signal of the pyrene moieties, and in particular of the excimer (Figure 4.16).
The excimer emission of XVII loaded in NPs was indeed strongly depleted by the addition of MDMA, cocaine and FMA. This can be attributed to the sterical hindrance of these drugs which, hosted in the cavity, prevent the formation of excimers or, at least, significantly decrease the probability of their formation, as envisaged in designing this chemosensor.
Noteworthy, MDMA has a much stronger effect than any other guest in depleting the excimer emission (Figure 4.17).
Figure 4.17 Fluorescence spectrum (λexc = 330 nm) of XVII + nanoparticles in H2O (c = 2x 10-6 M) upon addition of increasing amounts of MDMA (0 – 0,0025 M).
Furthermore, a dramatic decrease of the luminescence lifetime is observed both on the excimer and on the monomer emissions. Such observations prove the occurrence of an additional decay pathway that increases the rates of relaxation of both the excimer and monomer excited states. Such pathway can be attributed, since the energy transfer process from pyrene to MDMA is thermodynamically forbidden, to an efficient electron-transfer process involving the aromatic unit present in the MDMA molecule.
As far as the monomeric-type emission is concerned, we observed in all cases, as expected, an increase of the intensity of the monomeric emission, with a lower enhancement in case of sarcosine, and a subsequent decrease, after the initial steep increase, in case of MDMA (due to the onset of the electron-transfer quenching).
It is worth noticing that the presence of two different signals increase the possible information content that can be obtained from the analysis of fluorescence spectra, since the monomer-type emission responds to the presence in the analytic sample of this class of guests, while the decrease of the emission of the excimer-type emission can allow to discriminate the presence of MDMA among the other possible interferences.
Interestingly, the association constants that can be evaluated among the cavitand in the NPs (that can have, as previously described, a profound effect on the affinity for the analyte of a chemosensor) and the different species presents the following trend: MDMA > FMA > cocaine > amphetamine (Figure 4.18, sarcosine could not be evaluated due to the low signal variation). This trend is in agreement with the molecular recognition ability of the tetraphosphonate cavitand as demonstrated in a recent paper by isothermal titration calorimetry (ITC).39 Most important, cavitand XVII does not respond to sarcosine, which is strongly bonded by tetraphosphonate cavitands.40 These results strongly supports the proposed mechanism of detection, depicted in Figure 4.9.
Figure 4.18 Association constants in water solutions for the different analytes of XVII@NPs estimated from the titration curves reported in Figure 4.16.
81 All the results presented so far indicate that XVII can be an efficient chemosensor for this family of analytes in aqueous solution. In particular, the presence of MDMA can be clearly identified by looking at the intensity of the excimeric emission and, as additional signal, by the analyses of the excited state lifetime. In all other cases the presence of the drug can be identified looking at the increase of the intensity of the monomeric band, with possibility to use the excimer band, through a ratiometric approach, to avoid any calibration needs.