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pH-Sensitive Fluorescence Lifetime Molecular Probes Based on Functionalized Tristyrylbenzene Pedro J. Pacheco Liñán, Andres Garzon, Juan Tolosa, Ivan Bravo, Jesus CanalesVázquez, Julián Rodríguez-López, Jose Albaladejo, and Joaquin C. Garcia-Martinez J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b05526 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016

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The Journal of Physical Chemistry

pH-Sensitive Fluorescence Lifetime Molecular Probes Based on Functionalized Tristyrylbenzene Pedro J. Pacheco-Liñán,† Andrés Garzón,*,† Juan Tolosa,‡ Iván Bravo,† Jesús CanalesVázquez,§ Julián Rodríguez-López,|| José Albaladejo,⊥ and Joaquín C. GarcíaMartínez,*,‡ †

Departamento de Química Física, Facultad de Farmacia, Universidad de Castilla-La Mancha, Paseo de los Estudiantes s/n, 02071, Albacete, Spain. ‡ Área de Química Orgánica, Facultad de Farmacia, Universidad de Castilla-La Mancha, Paseo de los Estudiantes s/n, 02071, Albacete, Spain. § Instituto de Energías Renovables, Universidad de Castilla-La Mancha, Paseo de la Investigación 1, 02071, Albacete, Spain. || Área de Química Orgánica, Facultad de Ciencias y Tecnologías Químicas, Universidad de Castilla-La Mancha, Avenida Camilo José Cela 10, 13071, Ciudad Real, Spain. ⊥

Departamento de Química Física, Facultad de Ciencias y Tecnologías Químicas,

Universidad de Castilla-La Mancha, Avenida Camilo José Cela 10, 13071, Ciudad Real, Spain.

Abstract. The dependence of the fluorescence on pH for two 1,3,5-tristyrylbenzenes decorated with polyamine and poly(amidoamine) chains at the periphery was investigated. The highest fluorescence intensities were observed under acidic conditions because electrostatic repulsions between positively charged molecules reduce the fluorescence quenching. The slopes observed in the fluorescence pH-titration curves were associated with deprotonation of the different types of amine groups, which results in quenching by photoinduced electron transfer and aggregation processes. The linear dependence of fluorescence lifetime observed for different pH ranges is a valuable property for applications in the field of fluorescence lifetime sensors and imaging microscopy. The influence of the pH and the peripheral chains on the aggregation processes was also analyzed by absorption and emission spectroscopy, dynamic light 1 ACS Paragon Plus Environment


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scattering measurements and transmission electron microscopy. For compound 1, bands associated with the formation of aggregates were detected along with micrometric aggregates surrounded by fibers with lattice fringes typical of columnar mesophases. For compound 2, which contains longer peripheral chains with a higher degree of branching, aggregates with lower internal order were observed. In this case, the peripheral chains hindered aggregation by π-stacking although the amine groups did allow hydrogen bonding.

INTRODUCTION π-Conjugated molecules have attracted a great deal of attention in chemistry, biology and materials science due to their interesting optoelectronic properties and potential applications. As light emitters, both low molecular weight conjugated molecules and conjugated polymers are commonly employed in the construction of organic light emitting diodes (OLEDs),1-3 as fluorescent labels for biomolecules,4,5 and as analytical and biological sensors.6-8 In this context, numerous fluorescent conjugated molecules are being investigated as indicators of abnormal intracellular pH levels associated with diseases such as cancer and Alzheimer.7 As semiconductors, conjugated molecules are also used in organic field-effect transistors (OFETs) because of their good performance, light weight, flexibility and low cost in comparison with their inorganic counterparts.9-11 Nevertheless, many of the physical properties and applications of conjugated molecules strongly depend on self-assembly processes in which they form complex supramolecular structures.9,12,13 The appropriate functionalization of a fluorescent conjugated molecule can modify its physical properties and give rise to new and interesting applications. For example, 1,3,5-tristyrylbenzene is a light emitter that has been functionalized by García-Martínez

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and coworkers at the periphery to obtain dendritic architectures with tuned fluorescence responses.14 The combination of this molecule with flexible poly(amidoamine) (PAMAM) branches provided hybrid dendrimers that were able to transfect siRNA into neuronal cells15 and self-assemble into macromolecular structures that could stabilize small gold nanoparticles.16 The work described herein involved an investigation of the fluorescence intensity and lifetime dependence as a function of pH and protonation state of the different amine groups of two 1,3,5-tristyrylbenzene derivatives that bear either polyamine or PAMAM chains at the peripheral para positions (Chart 1). In addition to the fluorescence intensity, the dependence of fluorescence lifetime on pH is an interesting property for potential applications in time-resolved fluorescence imaging microscopy.8,17‒23 Fluorescence lifetime sensors are particularly promising for biological imaging techniques since lifetime does not depend on the fluorophore concentration, fluorescence intensity, excitation wavelength and duration of light exposure.8,20,21,23 The effect of the length and branching of the peripheral chains on the molecular selfassembly was also assessed by employing different spectroscopic and microscopic techniques. The understanding, control and optimization of the self-assembly of conjugated systems is one of the main challenges in the field of molecular electronics. Conjugated discotic molecules tend to stack one on top of another to form columns via π-π interactions.9 1D self-assembly depends on different factors such as the size and symmetry of the aromatic core, hydrogen bonding, metal coordination, and other interactions.9,12 Nevertheless, side-chain substituents can also control the morphology of 1D self-assembly, as described for different π-conjugated systems such as perylene diimides,24 hexabenzocoronenes,9,25 and triphenylbenzenes.26



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Chart 1. Structures of compounds 1 and 2.

MATERIAL AND METHODS Synthesis. Compounds 1 and 2 were obtained as reported previously.15 Briefly, the synthesis starts from a tristyrylbenzene trialdehyde core14 that provides the π-conjugated building block with optical absorption and emission properties. Reaction with an excess of ethylenediamine led to the corresponding triimine, which was reduced in situ with sodium borohydride to yield compound 1. The well-defined synthetic route to PAMAM dendrimers (Michael addition followed by amidation) was subsequently followed to prepare compound 2.15 Absorption and Fluorescence Measurements. UV-Vis absorption spectra were acquired on a Cary 100 (Varian) spectrophotometer at room temperature using a slit width of 0.4 nm and scan rate of 600 nm/min. Steady state fluorescence (SSF) and time resolved fluorescence (TRF) spectra were recorded on an FLS920 system (Edinburgh Instruments) equipped with a time correlated single photon counting (TCSPC) detector. A TLC 50 temperature-controlled cuvette holder (Quantum Northwest) was used for the measurements (temperature was controlled at 296 K). Quartz cuvettes (Hellma 4 ACS Paragon Plus Environment

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Analytics) of 10 mm were employed for all the spectroscopic measurements. For SSF spectra, a Xe lamp of 450 W was used as the light source and the excitation and emission slits were both fixed at 1 nm. The step and dwell time were 1 nm and 0.1 s, respectively. A EPLED 290 sub-nanosecond pulsed light emitting diode (Edinburgh Photonics) was employed as the light source at 291 nm for TRF experiments and the fluorescence decay profiles were collected at the wavelength of maximum emission. Aqueous solutions were prepared with a sample concentration of 1 µM and they were titrated by the successive addition of small volumes (in the order of microliters) of HCl and NaOH solutions of different concentrations (0.01–10 M) to an initial volume of 20 mL in order to minimize changes in the sample. In additional experiments, different samples with concentrations 0.5–5.0 µM, and containing diverse ions were also prepared to examine the formation of aggregates. The fluorescence intensity decay, I(t), was fitted to the following multiexponential function using an iterative least squares fit method = ∑ exp −/

(1)

where αi and τi are the amplitude and lifetime for each ith term. The mean lifetime of the decay was then calculated as =

∑

(2)

∑

The quantum yields of the samples, ϕx, were measured in solutions in 0.1 M HCl employing quinine sulfate (ϕs = 0.546) as the standard and applying the equation !" #

= !"

#

(3)

where I is the integrated density and the subscripts x and s correspond to the sample and standard, respectively. OD is the optical density and η is the refractive index, which is the same for sample and standard solutions.27 The step, excitation and emission slits were all fixed at 1 nm, dwell at 0.2 s and measured OD values were always lower than 5 ACS Paragon Plus Environment


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0.10. The relative quantum yield as a function of pH was, in turn, analyzed by means of eqn. (4) $% = &

'(

!")

)

!"'(

(4)

where the subscripts 0 and x(pH) correspond to the reference values measured for the sample in 0.1 M HCl solution and the values measured for the sample at different pH values, respectively. Dynamic Light Scattering (DLS) Measurements. DLS measurements were carried out on a Zetasizer NanoZS (Malvern Instruments). Aqueous solutions (20 mL) were prepared with a sample concentration of 4.0 µM and pH 2 by adding a concentrated HCl solution. The solutions were then passed through a nitrocellulose 0.22 µm pore-size filter. Particle size distribution was measured at different times before and after the addition of 100 µL of a concentrated solution of NaOH (3 M), with the final pH being about 12. Transmission Electron Microscopy (TEM). TEM studies were performed using a 2100 JEM (JEOL) electron microscope operating at 200 kV. Specimens for TEM observation were prepared by depositing aqueous solutions of the samples onto a holey carbon grid (EMS). Two solutions at pH 2 and pH 12 and a sample concentration ≤ 10–6 M were examined for each compound.

RESULTS AND DISCUSSION Spectroscopic Characterization. The optical properties of compounds 1 and 2 were investigated and the data obtained are summarized in Table 1 [see Figure S1 in the Supporting Information (SI) for more details]. The meta arrangement through which the three styryl units are linked to the central benzene ring prevents efficient delocalization and, as a result, the absorption maxima remained in the UV region. In all cases, the


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excitation spectra closely matched the respective absorption spectra. Both compounds were fluorescent and emitted blue light when irradiated – a typical response for stilbenoid compounds. Although contributions from more than one fluorescent species cannot be ruled out, it is assumed that in the emission spectra only the lowest energy π,π* transition was fluorescent. Significant changes were not observed in the excitation and fluorescence spectra of the two compounds and this is not unexpected given that the chromophore unit is identical and the electronic properties of the branches are similar. We previously showed that the maximum emission wavelength of this kind of compound strongly depends on the peripheral substitution.28,29

Table 1. Excitation and emission maximum wavelengths and quantum yields for compounds 1 and 2 in 0.1 M HCl at 296 K. absorption fluorescencea excitationb ϕFc compound λmax (nm) λmax (nm) λmax (nm) d 317 [~395(sh)], 413 315 0.08 1 d 319 396, 414 318 0.23 2 a Excitation at λmax of absorption. b Excitation spectra were collected with detection at λmax of fluorescence. c Fluorescence quantum yield determined relative to quinine sulfate in 0.5 M H2SO4 (ϕF = 0.546) as standard. d Two peaks can be distinguished in the emission band. (sh) corresponds to a shoulder. pH-Dependent Fluorescence. It can be seen from Figures 1a and S2 that the fluorescence intensity of the compounds is sensitive to pH, with the highest quantum yield achieved in acidic solutions. This pH-dependence in conjugated molecules is an interesting feature for applications in the field of fluorescent sensors.7,30-32 Fluorescence in hyperbranched materials has previously been observed in polymers and dendrimers due to the formation of emitting moieties by oxidation of internal tertiary amines under acidic conditions,33-36 but significant changes were not observed in the shape of the spectra at different pH values, except for those shifts related to aggregate formation, 7 ACS Paragon Plus Environment


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thus ruling out the formation of new fluorescence-emitting moieties by chemical reactions. Therefore, pH-dependent fluorescence must be due to physical quenching processes such as collisional deactivation, aggregation, and interactions between the tristyrylbenzene unit and the pendant groups.27 In this sense, previous studies on PAMAM dendrimers functionalized with fluorescent probes revealed that photoinduced electron-transfer (PET) from tertiary amines to the excited-state of the conjugated moiety is an efficient quenching mechanism.37-40 Non-protonated secondary and primary amine groups can also be efficient quenchers of polynuclear aromatic hydrocarbons by PET. Indeed, the bimolecular quenching constant for the PET interaction of 1,4-dihydroxy-9,10-anthraquinone with n-butylamine (0.57 × 1010 L mol– 1 –1

s ) is only 2.3 times lower than with tri-n-butylamine (1.32 × 1010 L mol–1 s–1).27,41


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a) 5

pH 1.9 pH 2.0 pH 2.1 pH 2.3 pH 2.6 pH 2.9 pH 3.1 pH 3.3 pH 3.9 pH 4.6 pH 5.2 pH 5.8 pH 9.4

7

Fluorescence intensity / 10 a.u.

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4

3

2

1

0 360

380

400

420

440

460

480

500

Wavelength / nm

b)

Figure 1. (a) Steady-state fluorescence emission spectrum of 2 as a function of the pH (c = 1 µM, λexc = 318 nm). (b) Fluorescence emission decays of 2 as a function of the pH (c = 1 µM, λexc = 291 nm, λem = 414 nm).

The relative fluorescence quantum yield as a function of pH is shown in Figure 2. Under acidic conditions protonation of the amine groups leads to elongated and ‘open’ configurations in lower-generation PAMAM or poly(propylene amine) 9 ACS Paragon Plus Environment


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dendrimers.42-43 Indeed, we have previously demonstrated that G4 PAMAM dendrimers decorated with polystyrylbenzene units at the periphery show enhanced fluorescence upon acidification because the open configuration of the PAMAM structure results in a decrease in the interaction between the chromophoric units and a reduction of the quenching process.40 Therefore, quenching must be minimized because the electrostatic repulsion between positively charged molecules must reduce the collisions and hinder the aggregation. In addition, protonation of the amines avoids PET processes.37-40 An increase in the branching at the peripheral chain can reduce the quenching effect at acidic pH and also provide a more hydrophobic environment, thus increasing the quantum yield.44-47 Therefore, under acidic conditions the highest ϕF value was determined for compound 2 (see Table 1). The increase in fluorescence quenching as a consequence of the deprotonation of the amine groups leads to ϕF values of less than 0.02 for both compounds under basic conditions.


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a)

c)

0.12

16.0

0.10

14.0 12.0 τm / ns

ϕ

0.08 0.06

10.0 8.0 6.0

0.04

4.0

0.02

2.0

0.00 0.0

2.0

4.0

6.0

8.0

10.0

0.0

12.0

1.0

pH

2.0

3.0

4.0

5.0 6.0 pH

7.0

8.0

9.0 10.0

d)

b) 0.24

16.0

0.20

14.0 12.0

τm / ns

0.16 ϕ

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0.12

10.0 8.0 6.0

0.08

4.0

0.04 2.0

0.00

0.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 pH

pH

Figure 2. Fluorescence quantum yields (ϕF) and mean emission lifetimes (τm) measured for 1 (a, c) and 2 (b, d) at different pH values.

In general, the titration curves for the mean emission lifetime (τm) showed similar profiles to those obtained for ϕF (see Figure 2). The small differences mainly correspond to variations in the UV-Vis absorbance spectra related to the formation of aggregated species that will be discussed in the next section. The highest τm values, which were in the range 14 to 16 ns, were determined at low pH and these dropped to < 6 ns in basic media. For both compounds, a linear dependence of τm with pH was observed in different pH ranges and the results of the corresponding fits are shown in Table 2. The linear dependence of τm with the concentration of an analyte is a valuable property for applications in the field of fluorescence lifetime sensors and imaging 11 ACS Paragon Plus Environment


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microscopy.8,17,18 Two linear pH ranges were observed for 1 and 2 and these could be related to the different amine groups present in the molecule and their chemical environments. It is known that the order of basicity of methylamines in solution is dimethylamine > methylamine > trimethylamine (reported pKa values at 298 K are 10.73, 10.66 and 9.80).48 Nevertheless, the proton affinity of the amine groups in the peripheral chains can be significantly influenced by features such as the proximity of other amine groups and their protonation state, the hydrophobic microenvironment, and the ionic strength of the medium. For instance, in a potentiometric pH titration study on PAMAM dendrimers, Niu et al. observed that the hydrophobic microenvironment within the dendrimer interior could reduce the proton affinity of a tertiary amine with respect to a similar amine outside the dendrimer (pKa was reduced by 1–2 pH units).49 The proton affinity of a tertiary amine can also be reduced due to the proximity of other protonated amine groups. In this sense, the lowest pKa values determined for PAMAM G0 by means of potentiometric titration were 3.15 and 6.68, corresponding to the protonation of the two internal tertiary amines that are linked by a two-carbon chain.50 In the next generation (PAMAM G1), pKa values in the range 3.07–7.10 were reported for the protonation of the six tertiary amines under the same experimental conditions.50

Table 2. Results of the linear fits for the different pH ranges in fluorescence lifetime titration curves. compound pH range slope (±2σ) intercept (±2σ) r2 4.4–5.9 –2.26 ± 0.34 25.47 ± 1.77 0.921 1 6.0–6.4 –5.91 ± 2.06 47.82 ± 12.72 0.892 2

1.3–3.2 5.7–7.2

–2.10 ± 0.12 –2.64 ± 0.24

17.45 ± 0.28 23.70 ± 1.56

0.990 0.941

In our case, compound 2 comprises nine external primary amines and six internal tertiary amines that can be classified into two groups depending on their chemical environment. In each branch, a two-carbon chain also links both types of 12 ACS Paragon Plus Environment

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tertiary amines. The experiments on the dependence of emission lifetime on pH (see Figures 1, 2 and S3) showed that the fluorescence quenching of 2 started at a pH as low as 1.3, with two slopes obtained in the titration curve. Two pKa values of 3.2 and 6.8 were calculated using the second derivative of the titration curve. These values are comparable to those mentioned above for the protonation of the two internal tertiary amines of PAMAM G0.50 Therefore, bearing in mind that the influence between tertiary amines of different branches should be small because of their considerable spatial separation, one may assume that each slope of the titration curve is associated with the deprotonation of a type of tertiary amine and its corresponding PET quenching, although quenching by aggregation induced by deprotonation of the tertiary amines cannot be totally ruled out due to the branched architecture of 2 and the possibility of multiple hydrogen bonds. The fluorescence lifetime quenching for compound 1 started at a significantly higher pH (pH > 4.4) due to the absence of tertiary amines. Two secant slopes can be observed in the titration curve. In this case, the assignment to a particular amine group is not trivial because primary and secondary amines have comparable proton affinities, which can be affected by the chemical environment and the protonation state of their neighboring amine. The pKa values reported for N-ethylethylenediamine, in which a primary amine is also linked to a secondary amine by a two-carbon chain, are 7.63 and 10.56.51 Therefore, deprotonation of only one type of amine must be observed within the studied pH range. As mentioned before, both PET and aggregation processes could cause fluorescence quenching. The smaller size of the peripheral chain in 1 might influence the nature of the aggregation, which could even proceed when some amines are protonated. In this context, the formation of aggregates in the presence of polyanions has been observed for methylene blue and pyrene moieties linked to



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PAMAM dendrimers.37,38 The continuous increase in the fluorescence quantum yield for 1 in the pH range from 2.1 to 4.3 could indicate premicellar aggregation or an enhanced fluorescence effect due to the formation of certain kinds of weakly coupled aggregates according to Exciton-Coupling Theory (see Figure 2a).44-46 The next section concerns the role of the peripheral chain and the structure of the aggregation, which is conditioned by distinct driving forces such as hydrogen bonds, π-stacking, and hydrophobic interactions.

Aggregation Measurements. From a qualitative point of view, the aggregation trend of the studied compounds in basic solution was corroborated by means of DLS measurements (Figures 3a and S4). In these experiments, polydisperse particles with Zaverage radius < 8 nm were measured at pH = 2 and these increased to reach stable values of several tens of nm after the addition of a small volume of a concentrated NaOH solution (final pH of the solutions was about 12). We previously reported the formation of micelle-like aggregates of 57 nm for 1 mM solutions of 2 at pH ~ 10, with a critical aggregation concentration of 9 ± 3 µM in water at pH = 8.2.16 Nevertheless, under more basic conditions (pH = 12) the formation of aggregates of 2 at even lower concentrations (4 µM) was observed by DLS measurements.


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Figure 3. (a) Evolution of the aggregation as a function of time before and after the addition of NaOH for compound 1 (c = 4 µM); (b) Normalized emission spectra of 1 at different pH values (c = 10 µM); (c-d) Absorption spectra of 1 at different pH values (c = 10 µM).

The emission band of compound 1 experienced a red shift and broadening at pH > 5 – behavior that was not observed for compound 2. At pH > 6 a new band centered at 497 nm, which could be related to the formation of aggregates, was observed (Figure 3b). In addition, the UV-Vis absorption spectra recorded for 1 in the pH range 1.1–5.4 showed two well-defined isosbestic points due to the decrease in the absorption band at 317 nm and the increase of the optical absorption at higher and lower wavelengths (Figure 3c). This trend seems to indicate the beginning of a premicellar aggregation



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process and matches the pH range in which the quantum yield of 1 increases. Two new isosbestic points and a peak at 255 nm were observed at pH values > 6.0 (Figure 3d). It is worth noting that the intersection point of the two straight lines of the titration curve obtained for 1 also correspond to pH ~ 6, which could indicate two pH regions with different aggregation states. The observed hypsochromic shift suggests the presence of H-type aggregates in which the molecular arrangement is face-to-face. Thus, the pronounced slope observed in the titration curve for 1 is consistent with the formation of strongly coupled H-aggregates, which in turn leads to strong quenching of the fluorescence and a shift of the emission band.52 Consequently, not only PET but also aggregation processes initiated by deprotonation of the amines seem to be responsible for the quenching. Blue shifts at higher pH values, which indicate the formation of Haggregates, have also been observed for different aromatic moieties linked to PAMAM branches.37,44,53 The higher length and branching degree of the peripheral chains in 2 may hinder the formation of ordered aggregates. In this sense, a decrease in the absorption was observed on increasing the pH, but neither a significant change in the spectrum shape nor a well-defined isosbestic point was clearly detected. Aggregation of 1,3,5-tristyrylbenzenes might cause toxicity problems in biological samples, limiting their use in fluorescence imaging applications. Nevertheless, the pH-sensitivity of compound 2 is mainly based in a PET mechanism rather than in ordered aggregation process and hence its application as fluorescence lifetime sensor cannot be totally ruled out.22 In this sense, the effect of the concentration, the ionic strength and the presence of metal ions on the fluorescence lifetime of 2 have been studied. In buffered solutions at pH 7.4, no clear dependence of τm on the concentration of 2 within the range 0.5‒5.0 µM was found (see Figure 4). Likewise, no significant effect on τm of 2 (in a concentration of 1 µM) was observed due


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to the presence of different salts (in mM concentrations) and Fe2+ ion (1 µM) (see Figure 4).

4

Fluorescence Lifetime / ns

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3

2

1

0

A

B

C

Figure 4. Fluorescence lifetimes measured for buffered solutions of 2 (Tris-HCl buffer, pH = 7.4) in presence of different salts. Samples A, B and C correspond to buffered solutions of 2 at the concentrations of 0.5, 1.0 and 5.0 µM. Different concentrations of salts were added to the sample B.

In TEM experiments, particles of 3.8 ± 1.0 and 3.8 ± 0.9 nm were observed for compounds 1 and 2, respectively, when the solutions were prepared in acidic media (pH = 2), as shown in Figures 5 and S5. These particle sizes could correspond to single molecules or small aggregates. As one would expect, more complex aggregations were observed for basic solutions (pH = 12) and these particles were in the order of micrometers and they were surrounded by fibers for compound 1. As can be seen in Figure 5b, these fibers are composed of strips with nanoscale dimensions and they exhibit a high degree of internal order, which is highly extended and seems to correspond to columnar mesophases formed by stacks of discotic molecules. These 17 ACS Paragon Plus Environment


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columnar mesophases have a periodicity of 0.34 ± 0.1 nm, which corresponds to a typical π-stacking distance, whereas lateral interactions between columns may result in the formation of extended three-dimensional networks.12 The formation of columnar mesophases is consistent with the formation of the H-aggregates observed by UV-Vis absorption spectroscopy. The longer peripheral chains in compound 2 hinder the π-stacking interactions and this results in the formation of entangled three-dimensional networks such as gel phases and lyotropic phases.12 Sporadic small aggregates (< 20 nm) that show some level of internal ordering, with a 0.26 ± 0.01 nm spacing between lattice fringes, were also found inside the micrometric aggregates composed of fibers with random orientations and without internal ordering (see Figures 5 and S5). Aggregates with short spacings between lattice fringes might be formed by hydrogen bonds involving the amines of the peripheral chains and not by π-stacking interactions. The lower internal order observed for aggregates of compound 2 compared to aggregates of 1 is consistent with the previously described DLS and UV-Vis absorption results.


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Figure 5. TEM images of (a) 1 from an acid solution, (b) 1 from a basic solution and (c) 2 from a basic solution.

CONCLUSIONS 19 ACS Paragon Plus Environment


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The dependence of the fluorescence intensity, quantum yield and lifetime with pH of two 1,3,5-tristyrylbenzenes substituted with polyamine and PAMAM chains has been studied. The pH dependence of the fluorescence was mainly attributed to quenching by PET and aggregation processes induced by deprotonation of the amine groups. Published pKa data for related structures were compared to our results in an effort to associate the different slopes observed in the fluorescence titration curves to deprotonation of the different types of amine groups. The linear dependence of τm with pH, which was observed for all of the compounds in different pH ranges, is an interesting property for possible applications in fluorescence lifetime sensors and imaging microscopy.8,17,18 The influences that pH and the size and chemical structure of the peripheral chains have on the aggregation processes were also studied by different analytical techniques. Particles of ≤ 0.5 nm corresponding to single molecules or small aggregates were observed for the studied compounds in acidic media by TEM, while more complex aggregations in the order of micrometers were detected under basic conditions. The formation of aggregates in solution at basic pH was also confirmed by DLS measurements. For compound 1, the aggregates observed by TEM are surrounded by fibers with lattice fringes with a periodicity 0.34 nm, which corresponds to a typical πstacking distance, while lateral interactions between columns can result in the formation of extended three-dimensional networks.12 In addition, bands corresponding to the formation of H-aggregates were detected in UV-Vis absorption spectra at pH ~ 6. Aggregates in the order of micrometers and surrounded by fibers with random orientations and without internal ordering were also observed for 2. Therefore, πstacking interactions seem to be the driving force for the formation of H-aggregates and columnar mesophases in compound 1. The longer length and higher degree of branching


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in the peripheral chains hinder the aggregation by stacking, although the amine groups allow hydrogen bonding and the formation of aggregates with lower internal order.

SUPPORTING INFORMATION Excitation and emission spectra of compounds 1 and 2 in 0.1 M HCl (Figure S1). Steady-state fluorescence emission spectra and emission decays recorded for 1 at different pH values for 1 (Figures S2 and S3). Nanoparticle size growth before and after adding a concentrated solution of NaOH to a solution of 2 (Figure S4). Additional TEM images of 1 and 2 (Figure S5).

AUTHOR INFORMATION Corresponding Authors *(A.G.) E-mail: [email protected] *(J.C.G.M.) E-mail: [email protected]

ACKNOWLEDGMENT The authors would like to thank the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha [FEDER projects, PEII11-0279-8538 and PEII2014-043-P (FOTOCINE)], Ministerio Español de Economía y Competitividad (CTQ2013-48411-P) and Universidad de Castilla-La Mancha (project GI20152964) for supporting the research described in this article.

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