2.1 Materials

Absolute ethanol was purchased from Carlo Erba Reagenti. Ultrapure water with a resistivity of 16 MΩ cm was produced using a Milli-Q apparatus (Millipore). The fluorescent dye 4-n-octylamino-7-nitrobenz-2-oxa-1,3-diazole (1) was prepared as described elsewhere [19], according to a variant of the synthesis reported by Heberer et al. [20]. PAA sodium salt (MW = 5100 g mol^–1) was purchased from Aldrich. Calf thymus DNA, “highly polymerized” sodium salt, was from Sigma.

2.2 Apparatus

UV/vis absorption spectra were recorded on a Hewlett–Packard 8452A diode array spectrophotometer. The measurements were conducted at 25 °C in a thermostatted cell. Corrected steady-state fluorescence spectra were registered with a Photon Technology International (PTI) Quanta Master 1 spectrofluorometer. The size and shape of the microcrystals were observed with a Zeiss MC80DX fluorescence microscope. The objective contained lenses of × 40 or × 100 magnification, the eyepiece had a lens of × 2.5 magnification. The microscope was equipped with a standard camera. The excitation wavelength was 430–450 nm, and the emission wavelength was set at around 500–530 nm, by the use of adequate filters. To prepare the samples, a droplet of the suspensions used for spectroscopic measurements was deposited on a slide after precipitation had been achieved.

3.1 Reprecipitation of 1 monitored by UV/vis spectroscopy

Compound 1 is hydrophobic, with a solubility limit as low as 4 × 10^–7 M in aqueous solution containing 2% ethanol (v/v) [19]. It is not ionized in this medium [20], although it is strongly polar due to the donor character of the amino substituent and to the withdrawing effect of both the furazan and the nitro groups.

For reprecipitation experiments, 40 μl of a dye solution at 10^–3 M in absolute ethanol was transferred to a quartz cell containing 1.96 ml of water, under continuous stirring. The final solution contained 2% ethanol (v/v) in water and 2 × 10^–5 M dye, which makes the dye concentration 50 times higher than the solubility limit. The reprecipitation process of 1 in water alone was described previously [14]. After introduction of the dye, the aqueous solution was yellow. Then it was progressively discolored as the organic compound precipitated, and red agglomerated microcrystals were deposited on the stirrer. The whole process, monitored by UV/vis absorption spectroscopy, took about 1 h.

In the present paper, reprecipitation was first carried out in the presence of anionic PAA (average MW = 5100 g mol^–1). The polymer was placed into water at a concentration of 5.9 × 10^–5 M, that is 3.2 × 10^–3 M in carboxylate groups. Then the ethanol solution of 1 was introduced, in the same conditions as before. The initial yellow solution was rapidly discolored, and the solution turned light pink. The reprecipitation process was drastically accelerated, since it was achieved within only 20 min. The UV/vis absorption spectrum showed strong variations (Fig. 2). Just after the introduction of the dye, the initial spectrum exhibited two bands. The first one, situated around 344 nm, corresponds to a π−π* transition, the second one with intense absorption at 476 nm, has been attributed to a charge transfer (CT) transition occurring between the donor and the acceptor groups of the molecule [21]. The position of these bands indicates that individual dye molecules are surrounded by an ethanol-rich environment. At the end of the reprecipitation process, the π−π* band and the CT band were, respectively, shifted to 364 and 496 nm, and their intensity was strongly decreased. Meanwhile, a new peak appeared at 546 nm. The baseline moved markedly upwards, due to the presence of particles in suspension that lead to scattering.

To study reprecipitation in the presence of calf thymus DNA, a stock solution was prepared containing approximately 1 mg DNA per ml water. The precise concentration of the DNA was determined from the UV/vis absorption spectrum, using ɛ²⁶⁰ = 13200 M^–1 cm^–1 for one calf thymus DNA base pair (bp) [22]. The stock solution was then diluted so that the DNA concentration was 5.0 × 10^–5 M bp. It can be noted that the DNA concentration was intentionally kept rather low, to prevent the medium from being too viscous. The concentrated solution of 1 in ethanol was added into it. The mixture lost its color twice as fast as when pure water was used. Almost not deposit was observed on the stirrer, and very small crystals, which made the solution blurred, precipitated slowly. When reprecipitation was complete, the UV/vis absorption spectrum showed three bands of weak intensity, with a maximum at 364, 496 and 546 nm, and a high baseline due to scattering. It is therefore very close to that of the suspension obtained in the presence of PAA.

3.2 Analysis of the suspensions by steady-state fluorescence spectroscopy

Steady-state fluorescence spectra were obtained on the suspensions after reprecipitation was completed. The total dye concentration was identical to before (2 × 10^–5 M). For the two types of suspension, the emission spectrum was strongly dependent on the excitation wavelength. Let us consider, for example, the suspension obtained in the presence of DNA. When excitation was performed at 482 nm, the emission spectra displayed a wide peak centered on 578 nm, and a shoulder at higher wavelengths, around 668 nm (Fig. 3). When excitation was performed at 510, then at 545 nm, the main emission band got narrower, while the long-wavelength emission band became progressively more intense. It must be noted that when exciting above 570 nm, where absorption is still significant, emission was no longer detected. This means that the transitions above 570 nm do not lead to an emissive excited state. Conversely, the excitation spectrum varied with the emission wavelength. When emission was set at 540 nm, the excitation spectrum only displayed a strong band peaking at 484 nm. For high wavelength emissions, the main band of the excitation spectrum moved slightly to the red, and a second band appeared, centered at 546 nm.

This behavior indicates the presence of several emitting species. One possibility is that these species are actually distinct populations of dye molecules, each experiencing a different environment. According to their location in the microcrystal, the strength of the interaction of the dye molecules with their neighbors may vary. The dye molecules may be in contact with water molecules or not. In organic solids, it is commonly accepted that emission arises from Frenkle-like excitons, that is excitation is localized on individual molecules. Stabilization depends on the interaction of the excited molecule with its crystalline surroundings [23].

For the sake of comparison, the excitation and emission spectrum of 1 dissolved in water with 2% ethanol was also given in Fig. 3. It must be noted that these spectra were recorded for a concentration of 1 higher than the solubility threshold, but in these conditions, precipitation is slow enough not to disturb the measurement. For the dissolved dye, the emission spectrum did not depend on the excitation wavelength and conversely, the excitation spectrum did not depend on emission, and was identical to the absorption spectrum. It can be noted that the emission spectrum of the dissolved dye is situated at shorter wavelengths than that of the microcrystal suspension. In contrast, the excitation spectrum of the dissolved dye was very close to the excitation spectrum of the microcrystals, when excitation was performed at 480 nm. This means that in the microcrystal suspension, some emitting molecules display the same spectral features as the dissolved dye, while other molecules show a totally different behavior.

3.3 Analysis by fluorescence microscopy

The suspensions were observed by fluorescence microscopy, at the end of the reprecipitation process. The particles grown in the presence of PAA (5.9 × 10^–5 M, that is 3.2 × 10^–3 M in carboxylate groups) were small rectangular structures displaying an X-shape pattern at the center. They were very flat and emitted yellow luminescence (Fig. 4, left). Three populations were distinguished, with sizes of about 40 × 15, 15 × 8, and 4 × 2 μm for the two largest sides. These particles are similar to the microcrystals grown in the presence of dendrimer. For the particles grown in the presence of DNA, two populations can be observed. Some are small rectangles (4 μm × 1 μm), emitting yellow light. Other rectangles were about 20 μm long. Their body emits green light, while orange–red light arises from the two small extremities (Fig. 4, right). This type of microcrystal had not been observed previously.