NRET — A rapid method to investigate the water–oil interface in reverse micellar systems

Journal of Molecular Liquids 214 (2016) 283–292

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NRET — A rapid method to investigate the water–oil interface in reverse micellar systems Gabriela Stîngă a, Monica Elisabeta Maxim a,⁎, Alina Iovescu a, Dan Eduard Mihăiescu b, Adriana Băran a, Anca Ruxandra Leontieş a, Marieta Balcan a, Dan Florin Anghel a a b

Colloids Chemistry Laboratory, “Ilie Murgulescu” Institute of Physical Chemistry, Romanian Academy, Spl. Independenţei 202, 060021 Bucharest, Romania Organic Chemistry Department, Faculty of Applied Chemistry and Materials Science, “Politehnica” University of Bucharest, 1–7 Polizu Street, 011061 Bucharest, Romania

a r t i c l e

i n f o

Article history: Received 10 December 2015 Accepted 19 December 2015 Available online 6 January 2016 Keywords: AOT/water/isooctane reverse micelle Non-radiative energy transfer Pyrene grafted poly(acrylic acid) Micellar interface Pyrene excimer

a b s t r a c t The water–oil interface of AOT/water/isooctane reverse micelles, with different hydration degrees (w0), is investigated by nonradiative energy transfer (NRET) between naphthalene (Np) probe and pyrene grafted poly(acrylic acid) (PAA150-Py32). The polymer is water-soluble while Np has affinity for the organic solvent. UV–Vis spectroscopy reveals that PAA150-Py32 is constrained in the micellar nanocage. The change of the pH of the micellar core and the addition of electrolyte are considered. FTIR and static fluorescence measurements confirm the presence of the polymer at the micellar interface. The stability and size distribution of the reverse micelles, with or without PAA150-Py32 and electrolyte are characterized by DLS. The photophysical parameters of PAA150-Py32 (the polarity index, I1/I3 and the polymer conformation index, IE/IM) are determined at different pHs and electrolyte concentrations. For w0 = 10 and w0 = 15, the pH does not influence the formation of Py excimers, which is an atypical behavior. NRET is strongly dependent on the pH, and for w0 = 10 and pH = 3.6 it reaches a maximum. The optimal salt concentration for which NRET is maximal is 5 × 10−4 M NaCl. The acquired results shed more light upon the stability of the micellar interface in various experimental conditions. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The reverse micelles of aerosol-OT (AOT) are thermodynamically stable systems, which can solubilize enzymes [1,2], proteins [3–5], polymers [6,7], dyes or natural pigments [8,9], and they are also used in the synthesis of organic or inorganic nanoparticles [10–12], etc. The size of micelles depends on hydration degree, which is the molar ratio between water and surfactant, w 0 = [H2 O]/[AOT] [1]. The water constrained inside the reverse micelles has raised much interest. To investigate its morphology and dynamics, several techniques like molecular dynamics simulation [13], mesoscopic simulation [14], ultrafast infrared spectroscopy [15–16], UV–VIS spectroscopy and fluorescence spectroscopy by means of probes [17–20] have been proposed. Various macromolecular species have been incorporated in AOT vesicles [21,22] or AOT/heptane/water microemulsions [7] in order to study their effect upon the stability, size or structure of the respective systems. The research of reverse microemulsions has reached new perspectives by the confining of fluorescent or fluorescently — labeled proteins inside those systems [23–25]. One knows that fluorescently labeled synthetic polymers offer additional information to that provided by the fluorescent probes [26] and their behavior is different from that of the fluorescent proteins. For hydrophilic polymers, the graft acts as a ⁎ Corresponding author. E-mail address: [email protected] (M.E. Maxim).

http://dx.doi.org/10.1016/j.molliq.2015.12.066 0167-7322/© 2015 Elsevier B.V. All rights reserved.

hydrophobic substituent, thus water soluble polymers labeled with fluorophores are seen as a special class of hydrophobically modified polymers [27]. The fluorophore, typically an aromatic hydrocarbon, is covalently attached on the macromolecular chain and gives information mainly about the conformation of the polymer statistical coil in the environments where it is hosted. At the same time the photophysical properties of the grafted polymer depends on the environment and changes under the action of external stimuli (pH, ionic strength, solvent, surfactant, another polymer, etc.) [26,28,29]. Pyrene (Py) is often used as a probe or graft in the investigation of regular micellar systems [30–33]. On the contrary, there is a lack of information on reverse micellar systems with fluorescently labeled polymers. The grafting of Py onto a pH-sensitive polyelectrolyte, like poly(acrylic acid) (PAA), allows tuning the macromolecular conformation and consequently, gives a more detailed report upon the hosting environment. This study aims to investigate AOT/water/oil systems by incorporating Py grafted poly(acrylic acid) (PAA150-Py32) as an internal sensor. This sensor provides information at nanometric level about the micellar water nanocage and its interface with the organic solvent. A big advantage comes from the high sensitivity of pyrene label to the polarity of the host environment. The action of external stimuli like pH and added electrolyte is systematically investigated. For several hydration degrees we check the nonradiative energy transfer (NRET) between the naphthalene probe dissolved in isooctane and the PAA150-Py32 solubilized in

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AOT micelles. The results provide information at nanoscale upon the structure and stability of the reverse micelle. This type of investigation may find application in testing the stability of various systems containing water–oil interfaces. An efficient energy transfer indicates a wellstructured and stable oil–water interface, reducing the time to acquire this kind of information. We find by NRET a rapid method for determining the micellar interface modification under the influence of certain stimuli like pH and electrolyte.

2. Materials and methods 2.1. Materials Sodium bis(2-ethylhexyl)sulfosuccinate (AOT, 98%) (Fig. 1A), pyrene (Py, ≥99%), naphthalene and isooctane, reagent grade, were purchased from Sigma-Aldrich, Germany. Sodium chloride, reagent grade, was obtained from Chimopar S.A., Romania. All the reagents were used as received. The working concentrations of AOT and naphthalene were 0.1 M and respectively 8.68 × 10−6 M. Poly(acrylic acid) (PAA) obtained from Wako Chemical, Japan, with MW = 150,000 gmol−1 was grafted with 3 mol% Py (Fig. 1B) and characterized as reported in a previous paper [27]. The grafted polymer has on average 1 Py graft for 32 monomeric acrylic units and therefore it will be noted as PAA150-Py32. The concentration of the grafted polymer in the experiments is 1.8 × 10− 4 M. The molar ratio of naphthalene probe/pyrene graft ([Np]/[Py] = 5) was calculated based on their molar extinction coefficients [32].

2.2. Methods 2.2.1. FTIR measurements FTIR measurements in reverse micellar systems were performed with a Nicolet i-S10 FTIR spectrometer, Thermo Scientific, equipped with cells with CaF2 windows and teflon spacers. The spectra were recorded in the 4000–400 cm−1 spectral region, and 32 scans at a resolution of 4 cm−1 were averaged to obtain each spectrum. For lyophilized PAA150-Py32, the spectrum was obtained by using the ATR (attenuated total reflection) device, equipped with diamond crystal and maintaining the same experimental parameters.

2.2.2. Measurements of dynamic light scattering (DLS) Measurements of dynamic light scattering (DLS) were done with a ZetaSizer, Nano ZS device from Malvern Instruments, England. The investigations were made at an angle of 173° and a laser wavelength of 633 nm.

2.2.3. UV–Vis absorption measurements UV–Vis absorption measurements were carried out with a Cary 100 Bio spectrophotometer from Varian, USA, equipped with quartz cuvette with pathlength of 1 cm. 2.2.4. The fluorescence measurements The fluorescence measurements were performed on a Fluoromax 4 spectrofluorimeter from Horiba Jobin Yvon, France. Pyrene emission spectra were recorded between 364 and 550 nm, for an excitation wavelength of 345 nm and with slits for excitation and emission of 3 respectively, 1 nm. The polymer conformation parameter (IE/IM) was calculated from the intensity ratio of the pyrene excimers, at 480 nm, to the pyrene monomers. The later intensity was calculated as the half-sum of the vibronic peaks intensities at 374 nm and 395 nm. The nonradiative energy transfer (NRET) is a very sensitive technique with nanometric precision which functions as a molecular ruler, because it can track phenomena that occur at distances between 1 and 10 nm [34]. NRET is a photophysical process by which an excited state fluorophore named donor (in our case naphthalene) transmits energy to a ground state fluorophore named acceptor (in our case pyrene) [35]. The phenomenon requires that the acceptor does not absorb energy at the wavelength at which the donor is excited and it takes place through dipole-dipole nonradiative coupling. NRET measurements were done at an excitation wavelength of 290 nm and the emission was recorded inbetween 310 and 550 nm. The slits for excitation and emission were respectively, 3 and 1 nm. 2.2.5. pH measurements pH measurements were done with Benchtop pH/ISE Meters 420 A, ATI ORION USA. Electrode calibration was performed using standard buffers of pH = 4 and pH = 7. All the samples were prepared with ultrapure water (18 MΩ cm resistivity) from Millipore Simplicity UV system device. The experiments were done at a constant temperature of 25 ± 0.1 °C. 3. Results and discussions 3.1. FTIR spectroscopy FTIR spectroscopy was used to characterize the types of water from reverse micelle and the chemical groups of the AOT/water/isooctane system in the presence of PAA150-Py32. IR spectrum of pyrene shows three intense bands at 841, 750 and 711 cm−1 [36]. For the lyophilized PAA150-Py32 one distinguishes the grafted pyrene peaks at 849, 799 and 711 cm−1 (Fig. 2A). They are assigned to C–H wagging vibration and confirm the presence of pyrene attached to PAA chain. The carbonyl of the amidic groups of PAA150-

Fig. 1. Chemical structure of: sodium bis(2-ethylhexyl)sulfosuccinate (AOT) (A); pyrene grafted poly(acrylic acid) (PAA150-Py32) (B).

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Py32 shows an absorption band at 1644 cm−1. The carboxyl group shows a strong absorption band (valence vibration of O-H) around 3000 cm−1. The absorption stretch band (valence vibration of C–H, ~ 2900, 2800 cm−1) from AOT spectrum partially overlaps that of the carboxyl group. The methylene (CH2) group has an absorption band at 1469 cm−1 and the methyl (CH3) group has an absorption at 1385 cm−1. In isooctane the absorption band of surfactant SO− 3 polar group (S = O symmetric stretching band) appears at 1050 cm−1 (spectrum not

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shown) and in AOT/water/isooctane reverse micelles it is shifted to lower frequencies, at 1047 cm−1 (being associated with a decrease in the force constant of S_O bond, spectrum not shown). The shift is caused by the interaction between water molecules and the SO− 3 group in the bound water inside the micellar core. When PAA150-Py32 is solubilized in the reverse micelles, one obtains the same value of the SO− 3 absorption peak (Fig. 2A). This indicates that the polymer does not interact with surfactant SO− 3 groups being embedded in the free water from the micellar core. For the ester group of AOT, the stretching band of the

Fig. 2. (A) FTIR spectra in the 600–4000 cm−1 stretch region for: 1. PAA150-Py32 and 2. AOT/water/isooctane/PAA150-Py32. (B) Original and FSD spectra of C_O in the 1700–1780 cm−1 stretch region for: 1. AOT/water/isooctane; 2. AOT/water/isooctane/PAA150-Py32; 3–7. AOT/water/isooctane/PAA150-Py32/NaCl with NaCl concentration: 2 × 10−4 M, 5 × 10−4 M, 1 × 10−3 M, 5 × 10−3 M, 9 × 10−3 M. (C) Original and FSD spectra of O–H in the 3100–3700 cm−1 stretch region for the same systems as at point B.

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Table 1 The average hydrodynamic diameter (dh) of AOT/water/isooctane/PAA150-Py32 micelles with or without NaCl, the relative intensity of the majority distribution (I) and the polydispersity index (PDI) of the sample. NaCl [M]

dh [nm]

I [%]

PDI

0M 2 × 10−4 M 5 × 10−4 M 7 × 10−4 M 1 × 10−3 M 5 × 10−3 M 7 × 10−3 M 1 × 10−2 M

11.36 10.91 10.75 10.18 10.12 9.123 7.612 7.660

94.9 90.8 87.3 88.8 90 100 92.9 94

0.209 0.219 0.237 0.223 0.214 0.143 0.250 0.189

C–O group appears at 1207 cm− 1. An asymmetric absorption peak of the C_O stretching vibration occurs at 1736 cm− 1 in the spectrum of AOT/water/isooctane, with or without polymer (Fig. 2B). By Fourier self deconvolution (FSD) of the spectra there are obtained two peaks (Fig. 2B): one at higher frequency (1739 cm−1), corresponding to carbonyl connected with C-1′ in AOT gauche conformation and one at lower frequency (1724 cm−1), corresponding to carbonyl connected with C-1′ in AOT anti-interleaved conformation. This agrees with results of Guowei et al. [37]. The water solubilized in AOT reverse micelles exists in four states: small amount of trapped water between the hydrocarbon chains of the surfactant in the micelle palisade, water bound through H bonds

by the sulphonyl groups, water bound by the sodium counterions and free water in the micellar core where there are no interactions between the surfactant and water molecules [14]. A strong and broad band of O–H stretching vibration (3100– 3700 cm − 1 ) provides information regarding the states of water constrained in reverse micelles and is processed by Fourier selfdeconvolution (FSD) to evidence the overlapping spectral features (Fig. 2C). For the AOT/water/isooctane micelle the maximum absorption of O–H band appears at 3455 cm−1, and by adding polymer and increasing NaCl concentrations one observes a shift toward higher frequencies (3463 cm−1). The spectra are processed by FSD and four appropriate peaks are obtained and presented below. For the AOT/ water/isooctane micelle the highest frequency peak is recorded at 3608 cm−1 and corresponds to the bound water in the palisade layer, lying around the hydrocarbon chains of the surfactant. By introducing the polymer and increasing electrolyte concentrations there is a slight shift to lower frequencies, at 3605 cm−1. The peak at 3502 cm−1 is assigned to the bound water which strongly interacts with the SO− 3 groups of AOT. By introducing the polymer in the system the peak shifts to lower frequencies, at 3495 cm−1 and with the addition of increasing electrolyte concentrations the peak appears at 3494 cm−1. The peak at 3411 cm−1 is assigned to free water from the micellar core where no interactions occur between the water molecules and the SO− 3 groups of AOT. By adding the polymer into the system the peak shifts to lower frequencies (3409 cm−1).

Fig. 3. The electronic spectra of the PAA150-Py32 in AOT/water/isooctane system, at various pHs (A) and at various NaCl concentrations (B); in water at different pHs (C). Wavelength of the maximum of absorption with the variation of pH and salt concentration for PAA150-Py32/water, PAA150-Py32/AOT/water/isooctane and PAA150-Py32/AOT/water/isooctane/ NaCl systems (D).

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Fig. 4. Emission spectra for PAA150-Py32 in AOT/water/isooctane systems at w0 = 5 (A); w0 = 7 (B); w0 = 10 (C); w0 = 15 (D).

The lowest frequency 3180 cm−1 corresponds to bound water due to its interactions with the sodium counterions of AOT. By adding PAA150-Py32 the peak shifts to higher frequencies, at 3188 cm−1, and with electrolyte addition there is a shift to 3190 cm−1, for 1 × 10−3 M NaCl. It can be assumed that the observed shift to higher frequencies (considering that the shift is in the opposite direction than the other three presented above), is due to the interaction of the amidic NH group of the PAA150-Py32 at the micelle interface.

3.2. DLS Dynamic light scattering measurements were carried out on AOT/ water/isooctane/PAA150-Py32 reverse micellar systems, with or without NaCl, to obtain information about the stability and size distribution of the studied micelles. An average hydrodynamic diameter (dh) of 10.80 nm was obtained for the AOT/water/isooctane micelle with w0 = 10. By adding PAA150-Py32 in the system, the value of dh increases at 11.36 nm which proves the polymer solubilization in the micellar core (Table 1). By adding increasing NaCl concentrations from 2 × 10− 4 M up to 1 × 10− 2 M, the average hydrodynamic diameter decreases to 7.66 nm. The data from Table 1 show a decrease of dh with salt addition up to 7 × 10− 3 M NaCl. For a subsequent increase in the electrolyte concentration dh remains constant. The decrease of the average hydrodynamic diameter by salt addition is due to the thinning of the electrical double layer of micelle, which was also observed by adding high ZrOCl2 concentrations in the AOT/water/isooctane system [38].

3.3. UV–Vis spectroscopy To investigate the influence of pH of aqueous polymer solution and of added salt we have performed UV–Vis absorption measurements on PAA150-Py32 solubilized in AOT/water/isooctane with w0 = 10. One may notice that the absorption peak appears at a constant wavelength of 344 nm (Fig. 3A). By NaCl addition in between (3 × 10− 4–7 × 10− 3) M and at constant pH, one observes that the salt has no influence upon the maxima of the absorption intensity which appear at the same wavelength of 344 nm (Fig. 3B). This phenomenon differs from what happens in aqueous solutions of PAA150-Py32 of increasing pH where a hypsochromic shift of the maximum occurs (Fig. 3C and D). These results indicate that the polymer chain solubilized in the micellar core is not as mobile as in aqueous solution, being constrained in the micellar nanocage.

3.4. Fluorescence spectroscopy 3.4.1. Photophysical behavior of PAA150-Py32 solubilized in AOT/water/ isooctane reverse micelles The properties of Py grafted on hydrophilic polymers differ from those of the Py probe due to the labels preassociation when the polymers are dissolved in water. Owing to their hydrophobic character the fluorescent labels affect the properties of the hydrophilic polymer even for low label contents. In our case, the emission spectrum of the Py label is characterized by four bands which appear at 374 nm (I1), 380 nm (I2), 385 nm (I3) and 395 nm (I5). The vibronic peak from 387 nm (I4) which appears

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for Py probe is not visible for the grafted polymer (see Fig. 4). Among these bands the first and the third are important because their ratio (I1/I3), known as the polarity index, gives information about the polarity of the medium in which Py is solubilized [30,39]. Fluorescence data on AOT/water/isooctane/PAA150-Py32 at various w0 and pH in between 2 and 9 (Fig. 4) allowed calculating the Py photophysical parameters, namely I1/I3 and IE/IM. Fig. 5A presents the variation of I1/I3 with the pH, in the environment sensed by Py labels. One observes that for all studied w0 and pH values, the Py grafts are localized in an environment with different polarity from that indentified by Py probe in water (I1/I3 = 1.87) or isooctane (I1/I3 = 0.59) [40]. For w0 = 5, 7, 10 the polarity index increases with the pH of the aqueous PAA150-Py32. The polymer chains are loosened and the pyrene grafts sense a medium with higher polarity as the pH turns to basic. For w0 = 5 the rise of I1/I3 is more pronounced, that is from 1.37 to 1.63. At w0 = 7, I1/I3 increases from 1.62 to 1.72, and at w0 = 10, I1/I3 increases from 1.70 to 1.74. By rising w0 and the pH of the micellar core the Py grafts gradually sense an aqueous-like environment which we assign to the free water inside the micelle. The systems with w0 N 11 are named microemulsions (W/O microemulsions) due to the existence in the micellar core of a bulklike water [41]. For w0 = 15 the ratio I1/I3 fluctuates in between 1.46 and 1.39 with pH variation, having a minimum at pH = 6.3. For this hydration degree the micellar core is greater than previously and allows a

better uncoiling of the polymer. However the values of the polarity indexes are smaller than for w0 = 7 and w0 = 10. The area of the surfactant polar groups increases with w0 [42] and the electrostatic repulsions between the polymer carboxylates and the surfactant sulfonates are stronger. The polymer chains will be forced to have a smaller uncoiling and thus, the Py grafts will be less exposed to the water from the micellar core. At w0 = 10, fixed pH and variable salt content (Fig. 5B), the polarity ratio is almost constant until 5 × 10−4 M NaCl, than it decreases up to 1 × 10−3 M wherefrom it stays on a plateau. Former DLS measurements indicated for PAA150-Py32 (10−2 M) in aqueous solution an average hydrodynamic diameter of 55 nm while in the presence of NaCl (10−2 M) it was considerably smaller (13 nm) [31] due to the screening of the polyelectrolyte charges. The added salt diminishes the diameters of the polymer coils reducing their size polydispersity. As the polymer coils are tightly winded the Py grafts are not exposed to the salt solution and sense a medium with lower polarity (I1/I3 = 1.56) than the micellar core (1.74) or bulk water (1.87). The changes of micropolarity (I1/I3 index) are very small in the studied range of NaCl concentrations but Py is still capable to sense them. Fig. 6A illustrates the effect of pH upon the conformational parameter of the grafted polymer in the micellar system AOT/water/isooctane/ PAA150-Py32/Np which also solubilizes Np. One could observe that for w0 = 5, IE/IM has a maximal value up to pH = 4, followed by a continuous descending until pH = 9. For a small w0 and an acid pH, the

Fig. 5. The polarity index I1/I3 of PAA150-Py32 in AOT/water/isooctane reverse micelles, as a function of pH (A) and NaCl concentration (B).

Fig. 6. The effect of pH upon the polymer IE/IM parameter in AOT/water/isooctane/ PAA150-Py32/Np systems (A). The effect of the added NaCl upon IE/IM (B).

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polymer coil is strait-laced in the micellar nanocage and the number of Py excimers is maximal because the distance among the Py grafts is small. At pH N 4 the polyelectrolyte begins to dissociate in a higher proportion, the polymer chains slightly relax and the distance between the Py grafts increases. Consequently, the number of excimers decreases until pH = 9. For the system with w0 = 7 the plateau of constant IE/IM sprawls until pH = 5, which is close to the value of the acidity constant (pKa = 5.1) of Py labeled poly(acrylic acid) (Mw = 150,000) in aqueous solution [43]. As the polymer dissociates the IE/IM values continuously decrease up to pH = 9. One has observed that for the systems with w0 = 5 and w0 = 7, as the micellar pH increases, IE/IM starts to decrease from a pH value close to the pKa of the polymer. This accords with the studies of Melo and collaborators which showed that for Py-grafted

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PAA in aqueous solution, IE/IM decreases with pH rising [44]. It was also shown that at low pH the associative behavior of the polymer predominates, while at high pH the electrostatic repulsions are predominant [43]. With pH rising the polymer chains relax as is permitted by the micellar core and the distance between grafts increases leading to a smaller number of excimers. In the systems with w0 = 10, IE/IM has small values and almost no variation with the pH. At w0 = 15 IE/IM has slightly higher values than for w0 = 10, but the same behavior with the pH variation. One observes that at w0 = 10 and w0 = 15 the pH has no obvious influence upon the excimers formation which is atypical and is owned firstly, to the electrostatic repulsions between the negative polar groups of the surfactant − (SO− 3 ) and the negative charges of the polymer (COO ) and secondly, to the engage of the Py grafts in the NRET. The involvement of the Py

Fig. 7. NRET in the AOT/water/isooctane/PAA150-Py32/Np systems with: w0 = 5 (A), w0 = 7 (B), w0 = 10 (C), w0 = 15 (D), w0 = 10 and added NaCl (E). The spectra are normalized to the Np emission at 319 nm.

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Fig. 8. The variation of IPy/INp with the pH in AOT/water/isooctane/PAA150-Py32/Np systems.

grafts in the nonradiative energy transfer with the Np probe, at the micellar interface, will be discussed in Section 3.4.2. By increasing the added NaCl in AOT/water/isooctane/PAA150Py32/Np, at w0 = 10 one observes an increase of IE/IM for 5 × 10−4 M NaCl which is followed by a plateau for the whole studied range of salt concentrations (Fig. 6B). This phenomenon is explained by a decrease in the size of the polymer coils with NaCl addition until a salt concentration over which the polymer no longer collapses. Concomitantly, the distance between the Py grafts diminishes and the number of excimers increases up to a value which remains constant on the whole range of salt concentrations. 3.4.2. NRET in AOT/water/isooctane reverse micelle NRET was used to investigate the micellar interface of the AOT/ water/isooctane/PAA150Py32/Np systems. To this aim the Np probe was excited at 290 nm and the emission of PAA150Py32 was registered inbetween 310 and 550 nm. The naphthalene introduced in the reverse micellar systems AOT/water/isooctane with various hydration degrees (w0 = 5, 7, 10, 15) has affinity for the nonpolar solvent. The Py grafted

polyelectrolyte PAA150-Py32 is solubilized in the aqueous core of the reverse micelles. In all studied systems the pH was varied between 2 and 9. For AOT/water/isooctane with w0 = 10 the concentration of added electrolyte was inbetween 3 × 10− 4–7 × 10− 3 M and did not affect significantly the original pH of the polymer. The systems with w0 = 5, 7, 10, 15 without electrolyte and those with w0 = 10 and added electrolyte present an emission from 310 to 360 nm. This is due to the locally excited Np which has the (0,0) band at 319 nm (Fig. 7). The naphthalene emission has only two distinct peaks, at 319 and 334 nm. The Py emission is dependent on pH and occurs after 360 nm. The emission of Py excimers is not structured and has a maximum around 480 nm. Py emission occurs by energy transfer from the excited Np and one may notice that NRET takes place for all studied pH values. To measure the NRET efficiency between the Np probe and the Py labels one calculates the IPy/INp parameter which represents the ratio between the emission intensity of the Py monomer at 395 nm and the emission intensity of Np at 319 nm. Fig. 8 illustrates the evolution of IPy/INp as a function of the pH of the aqueous solution from the micellar core. One has observed that for a low hydration degree w0 = 5, NRET is very small and is not influenced by the pH. The IPy/INp values describe a plateau of constant values (0.59–0.60). The polymer coil is strait laced in the aqueous micellar core of small dimension and the Py grafts are not exposed to the interface to be able to interact with the Np probes. For w0 = 7, IPy/INp presents a maximum at pH = 3.7 (IPy/INp = 1.24) and a plateau of constant values above pH = 5. For w0 = 10, IPy/INp has higher values than for the other hydration degrees. From pH 2.3 till pH 3.6 (IPy/INp = 2.72) one observes a sharp increase of IPy/INp followed by a decrease up to pH = 4 and then by a flat region. At pH = 2.3, PAA150-Py32 has a small dissociation degree, with a low number of carboxylate groups. The electrostatic repulsions between the polar groups of AOT and the polyelectrolyte carboxylates are very small allowing naphthalene and pyrene to get closer. This phenomenon occurs also at pH = 3.6 for which a maximal NRET is registered, at a polymer dissociation degree of 2.87%, determined in agreement with Cesarano III and collaborators [45] for a pKa ~ 5.1 estimated by Costa and Melo [43]. The flat region between pH 4 and 9 may be explained by the increase of the dissociation degree when the polymer gets many more negative charges and the statistic coil unwinds adopting an extended conformation. At high pH, as the dissociation degree is

Fig. 9. AOT/water/isooctane reverse micelle and schematically NRET between Np probe and Py graft.

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elevated, the electrostatic repulsions raise and the Py grafts are maintained at a certain distance from the Np probes. Consequently, in between pH = 5–9 the NRET among the two fluorophores is almost constant. The obtained results indicate that w0 = 10 and pH = 3.6 are the most favorable conditions which allow the Py grafts to localize at the micellar interface close to Np (dissolved in the organic solvent) for an optimum NRET. For the systems with w0 = 15, IPy/INp decreases from pH = 2.6 (IPy/INp = 1.75) until pH = 5.4 where a minimum is reached (0.84) then a slightly increase occurs up to pH = 9 (IPy/INp = 1.03) as the polymer coil relaxes. Though at small pH the polyelectrolyte is very little dissociated the values of IPy/INp decrease as the pH rises. This phenomenon differs from what happens at the other hydration degrees. The headgroups surface area of AOT increases with w0 [42] and therefore the polyelectrolyte penetrates no more the micellar interface as it does at w0 = 10, due to the stronger electrostatic repulsions between the surfactant SO− 3 groups and the polymer carboxylates. Consequently, the NRET efficiency is smaller. On the other side, the experimental results lead to the conclusion that at high hydration degrees the pairs Np — grafted Py have a similar behavior with that obtained in AOT direct micelles formerly investigated [31], where PAA150-Py32 is solubilized in water and Np in the hydrophobic micellar core. Fig. 9 schematically describes the AOT reverse micelle and the NRET phenomenon at the micellar interface. The electrolyte addition into the micellar core modifies the behavior of both polyelectrolyte and AOT reverse micelle and implicitly the NRET among the two fluorophores. The increase of salt concentration strongly screens the electrostatic repulsions between the electric charges of the polyelectrolyte. At the same time, the salt reduces the electrostatic repulsion between the surfactant polar groups SO− 3 and polyelectrolyte COO− groups. These conditions facilitate the approaching between the two species involved in the nonradiative energy transfer. The value of optimum NRET, I Py/INp = 1.56 is obtained for 5 × 10 − 4 M NaCl (Fig 10). The descending region of the curve presented in Fig. 10 shows that by increasing the NaCl concentration, the amount of solubilized water decreases due to the “salting out” effect of the electrolyte. Vasquez and Williams affirm that by increasing the salt (ZrOCl2) concentration in the micellar core of AOT/water/isooctane reverse micelle, the system loses its stability due to the electrolyte hydration, which diminishes the solubilized water from the micellar core [46]. Consequently, the amount of solubilized PAA150-Py32 is reduced and the NRET decreases.

Fig. 10. Variation of IPy/INp with NaCl concentration in the AOT/water/isooctane/PAA150Py32/Np system at constant pH.

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4. Conclusions The pyrene grafted poly(acrylic acid) senses a restricted environment in reverse micelles of AOT/water/isooctane, with or without added electrolyte. UV–Vis and DLS measurements demonstrate the polymer solubilization in the micellar nanocage. FTIR and fluorescence results are correlated with the polymer presence at the micellar interface. The study provides information about the polarity of the environment sensed by the Py grafts, the conformation of the polymer strait-laced in the micellar core and the NRET occurring among naphthalene probe and pyrenes. The emission of Py strongly depends on pH and NRET occurs for all studied pH values. The data indicate that for w0 = 10 and pH = 3.6, the Py grafts efficiently penetrate the micellar interface where Np probes are localized and an optimal NRET occurs among the two fluorophores. At salt addition, NRET is maximal for 5 × 10− 4 M NaCl. An efficient energy transfer in systems of this type is obviously an indicative for a stable oil–water interface. Therefore, the obtained results recommend the grafted polymer for a quick appreciation, via NRET, of the interfacial stability in reverse micellar systems with industrial and biomedical application. Acknowledgments This work has been financially supported by the Romanian Academy within the research program “Functional Complex Colloids” of the “Ilie Murgulescu” Institute of Physical Chemistry. The authors gratefully acknowledge the support of EU (ERDF) and Romanian Government allowing for acquisition of the research infrastructure under POS-CCE O2.2.1 project INFRANANOCHEM, No. 19/2009.03.01 and the support from PN-II-ID-PCE-2011-3-0916 grant. The contribution of Dan Eduard Mihăiescu has been funded by the Sectoral Operational Programme Human Resources Development 2007–2013 of the Ministry of European Funds through the Financial Agreement POSDRU/159/1.5/S/132397. References [1] R. Biswas, S.K. Pal, Caging enzyme function: α-chymotrypsin in reverse micelle, Chem. Phys. Lett. 387 (2004) 221–226. [2] P. Majumder, R. Sarkar, A.K. Shaw, A. Chakraborty, S.K. Pal, Ultrafast dynamics in a nanocage of enzymes: solvation and fluorescence resonance energy transfer in reverse micelles, J. Colloid Interface Sci. 290 (2005) 462–474. [3] A.V. Martinez, S.C. DeSensi, L. Dominguez, E. Rivera, J.E. Straub, Protein folding in a reverse micelle environment: the role of confinement and dehydration, J. Chem. Phys. 134 (2011) 055107. [4] D. Wu, R. Li, D. Fan, Y. Zhang, Q. Wei, Sensitive determination of protein using terbium — metalloporphyrin as a fluorescent probe in AOT microemulsion, J. Mol. Liq. 199 (2014) 67–70. [5] J.M. Kielec, K.G. Valentine, A.J. Wand, A method for solution NMR structural studies of large integral membrane proteins: reverse micelle encapsulation, Biochim. Biophys. Acta 1798 (2010) 150–160. [6] S.K. Mehta, S. Sharma, Temperature-induced percolation behavior of AOT reverse micelles affected by poly(ethylene glycol)s, J. Colloid Interface Sci. 296 (2006) 690–699. [7] T.H. Wines, P. Somasundaran, N.J. Turo, S. Jockusch, M.F. Ottaviani, Investigation of the mobility of amphiphilic polymer-AOT reverse microemulsion systems using electron spin resonance, J. Colloid Interface Sci. 285 (2005) 318–325. [8] J.A. Gutierrez, R.D. Falcone, J.J. Silber, N.M. Correa, C343 behavior in benzene/AOT reverse micelles. The role of the dye solubilization in the non-polar organic pseudophase, Dyes Pigments 95 (2012) 290–295. [9] H.-R. Park, S.-E. Im, J.-J. Seo, K.-M. Bark, Spectroscopic properties of quercetin in AOT reverse micelles, Bull. Kor. Chem. Soc. 35 (3) (2014) 828–832. [10] D. Singha, N. Barman, K. Sahu, A facile synthesis of high optical quality silver nanoparticles by ascorbic acid reduction in reverse micelles at room temperature, J. Colloid Interface Sci. 413 (2014) 37–42. [11] M.G. Spirin, S.B. Brichkin, V.F. Razumov, Growth kinetics for AgI nanoparticles in AOT reverse micelles: effect of molecular length of hydrocarbon solvents, J. Colloid Interface Sci. 326 (2008) 117–120. [12] K. Naoe, S. Yoshimoto, N. Naito, M. Kawagoe, M. Imai, Preparation of protein nanoparticles using AOT reverse micelles, Biochem. Eng. J. 55 (2011) 140–143. [13] D.E. Rosenfeld, C.A. Schmuttenmaer, Dynamics of water confined within reverse micelles, J. Phys. Chem. B 110 (2006) 14304–14312. [14] S.L. Yuan, G.W. Zhou, G.Y. Xu, G.Z. Li, Investigations of water morphology in reverse micelles: mesoscopic simulation and IR spectral analysis, J. Dispers. Sci. Technol. 25 (6) (2004) 733–739.

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