1. Introduction

Two-photon absorption (2PA) is characterized by the simultaneous absorption of two photons and therefore increases with the square of the light intensity. This nonlinear effect has substantial advantages over conventional one-photon absorption and can be utilized for many applications such as microfabrication, optical power limiting, three-dimensional optical data storage and bioimaging [1]. Typically, near infrared light is used for excitation of organic molecules showing 2PA.

Biological tissues show high transmission at longer wavelengths enabling deeper imaging of samples. The low foot print of two-photon fluorescence in the sample as well as the low energy of wavelengths used to effect the two-photon absorption minimizes photodamage. This facilitates in vivo monitoring of alive biological samples, an attribute much valued in translational cancer research [1]. Though high laser intensities are required to induce two-photon fluorescence, the nonlinear nature of this phenomenon limits it to the confines of the laser focus. The fluorescence diminishes rapidly with decrease in intensity of exciting radiation at laser focus. This leads to higher resolutions in two-photon fluorescence imaging [1].

The design and synthesis of efficient 2PA dyes with strong two-photon excited fluorescence has been intensively investigated to develop tailor-made 2PA materials. A large 2PA cross-section (TPACS) can be obtained by using π-electron donor-acceptor-donor (D-A-D) structures with a highly conjugated π-system [1, 2]. Additionally, high fluorescence under physiological conditions is required for biophotonic applications. However, many 2PA molecules are only soluble in organic solvents and suffer from fluorescence quenching in highly concentrated solutions or when aggregating. Aggregation induced fluorescent dyes are a class of dyes which shows high fluorescence on aggregation and thus overcoming the drawbacks of conventional dyes [2]. Fluorescence behavior is affected by the solvent polarity and the local environment and depends on the molecular structure of the dyes in solution and aggregated state [3]. Therefore, molecules which are capable of specific interactions, such as J-aggregation or excimer formation, tend to show aggregation enhanced fluorescence which is particularly interesting for gaining strong signals in biophotonic applications.

In this paper, we report the synthesis and characterization of a 2PA dye based on a 3,3-(thiophene-2,5-diyl)bis(2-(4-bromphenyl)acrylonitrile) (Tp-bis(PhBr-ACN)) core using triphenylamine (TPA) as electron-donor moiety. Symmetrical D-A-D structures similar to our 2PA dye are known to show a large two photon absorption cross section due to the highly extended π-conjugation and the likelihood of an internal charge transfer (ICT) [4]. Linear and non-linear optical properties, solvatochromism and aggregation effects of Tp-bis(StACN-TPA) have been investigated.

2. Experimental

2.1. Materials

All starting materials were purchased from Sigma-Aldrich or Tokyo Chemical Industry. All reagents purchased commercially were used without any further purification if not otherwise mentioned. Tp-bis(PhBr-ACN) was kindly provided by Prof. H.-K. Shim’s lab in KAIST. All solvents used for column chromatography were purchased from Samchun Pure Chemical.

2.2. Instruments

¹H and ¹³C nuclear magnetic resonance (NMR) spectra and COSY and HSQC spectra were recorded using a Bruker AVANCE II spectrometer operated at 500 MHz (125 MHz respectively for ¹³C). ¹H NMR spectra at 300 MHz were recorded using a Varian Mercury NMR spectrometer. For internal reference tetramethylsilane was used. UV-visible spectra and photoluminescence spectra were measured using a Shimadzu UV-3600 UV-Vis-NIR spectrophotometer and a Scinco FluoroMate FS-2 fluorescence spectrometer, respectively. IR measurements were carried out using a Shimadzu IR-Affinity-1S FT-IR spectrophotometer using an ATR-Unit. Matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) spectra were recorded using a Voyager-DETM STR Biospectrometry Workstation model using a dithranol matrix. The two-photon absorption cross-section (TPACS) was obtained by two-photon induced fluorescence method using a Ti-sapphire 100 femto-second pulse laser with a repetition rate of 90 MHz. Rhodamine 6G was used as reference fluorophore for the TPACS measurement.

2.3. Synthesis of 4-(diphenylamino)benzaldehyde (TPA-CHO)

TPA-CHO was synthesized by Vielsmeyer-Haack reaction according to literature [5]. A dry 50 mL Schlenk-Flask under N₂ atmosphere was charged with 1.77 mL (1.68 g, 23.0 mmol, 2.8 eq) N,N-dimethylformamide (DMF). While cooling with an ice-water bath, 2.12 mL (3.56 g, 23.2 mol, 2.8 eq) freshly distilled POCl₃ were added dropwise. After that, the water-ice bath was removed and 2.00 g (8.2 mol, 1 eq) triphenylamine were added as a solution in 25 mL DMF. The flask was sealed using a septum and N₂ balloon and heated for 34 h at 40 °C. The solution was cooled to room temperature and poured into 100 mL water. After cooling the mixture for 5 h at −18 °C the yellow precipitate was filtrated using vacuum filtration. The product was purified by column chromatography using n-hexane and ethyl acetate (19:1) as eluent to obtain 1.50 g (5.5 mmol, 67 %) of a yellowish solid.

2.4. Synthesis of N’N-diphenyl-4-vinylaniline (TPA-C=C)

TPA-C=C was synthesized by Wittig olefination of TPA-CHO [6, 7]. A 25 mL Schlenk flask was freed of water and oxygen using Schlenk technique. While maintaining a constant N₂ stream 696 mg (1.95 mmol, 2.7 eq) methyltriphenylphosphoniumbromide (CH₃PPh₃Br) was added to 5 mL THF (freshly dried and distilled over sodium). The flask was cooled to 0 °C using an ice water bath and 175.2 mg (7.3 mmol, 10 eq) sodium hydride were added slowly. The cooling bath was removed and the mixture was stirred until the white suspension turned green (ca. 30 min at RT). After that 200 mg (0.73 mmol, 1 eq) TPA-CHO were added to the greenish suspension which turned instantly grayish. The flask was sealed using a septum and a N₂ balloon and stirred for 24 h at room temperature. The reaction mixture was poured slowly into 100 mL water-ice (1:1 ratio) and extracted three times with 100 mL methylene chloride. The combined organic phase was washed using 25 mL brine and dried over MgSO₄. After evaporation of the solvent the crude product was purified using column chromatography (n-hexane:ethyl acetate = 19:1) to obtain 178.8 mg (0.66 mmol, 90 %) as a yellow solid showing blue PL when excited at 365 nm UV light.

2.5. Synthesis of (2Z,2’Z)-3,3’-(thiophene-2,5-diyl)bis(2-(4-((E)-4-(diphenylamino)styryl) phenyl)acrylonitrile) (Tp-bis(StACN-TPA))

Tp-bis(StACN-TPA) was synthesized by Heck coupling of TPA-C=C with (2Z,2’Z)-3,3’-(thiophene-2,5-diyl)bis(2-(4-bromophenyl)acrylonitrile) (Tp-bis(BrPhACN)) [8]. While maintaining a constant N₂ stream 54.8 mg (0.201 mmol, 2.1 eq) TPA-C=C, 47.8 mg (0.096 mmol, 1 eq) Tp-bis(BrPhACN), 10.5 mg (0.035 mmol, 0.35 eq) tri(o-tolyl)phosphine and 1.8 mg (0.008 mmol, 0.08 eq) Pd(OAc)₂ were added to 2.1 mL DMF (freshly dried and distilled over CaH₂) and 0.24 mL trimethylamine (freshly dried and distilled over CaH₂) in a dried 25 mL Schlenk flask. The mixture was degassed three times using freeze-pump-thaw cycling and heated to 140 °C under static vacuum. After the color changed from yellow to dark-red (ca. 1 h) the mixture was heated for further 48 h and after that cooled to room temperature. The mixture was poured into 200 mL water and extracted 4 times with 100 mL methylene chloride (MC). The combined organic phase was washed with 50 mL brine and dried over MgSO₄. The crude product was purified using gradient column chromatography starting with n-hexane:MC = 7:3 slowly increasing the amount of MC until pure MC was used for elution. The product TP-bis(StACN-TPA) was obtained as a dark red shiny solid at a yield of 44 %, 37 mg (0.042 mmol).

2.6. Preparation of nanoaggregates

Nanoaggregates were prepared by a simple precipitation method using 50 µL of a 0.1 mg/mL concentrated stock solution of Tp-bis(StACN-TPA) in THF. The aliquots were diluted to a total volume of 4 mL starting with THF and slowly adding water. Solutions from 10 to 100 % ratio of THF with a final concentration of 6.25 µg/mL Tp-bis(StACN-TPA) were produced by this method.

3. Results and discussion

3.1. Synthesis

The synthetic route for Tp-bis(StACN-TPA) is shown in Fig. 1. For the detailed procedure please refer to experimental part. In the final symmetrical product the triphenylamine-groups act as electron-donor (D) moieties while the thiophene-bis-acrylnitrile-group acts as an electron-acceptor (A) moiety forming a symmetrical D-A-D structure. The highly aromatic dye shows good solubility in low to medium polarity solvents like toluene, diethyl ether, ethyl acetate, methylene chloride and acetone. It is also soluble in THF which can be used for the formation of nanoaggregates by water addition. In polar solvents like DMSO or DMF the solubility was limited.

 

Fig. 1 Synthetic scheme of Tp-bis(StACN-TPA).

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3.2. Linear optical properties

The linear optical properties of Tp-bis(StACN-TPA) were investigated using UV-Vis absorption and photoluminescence (PL) measurements in solvents with different polarities at various concentrations as listed in Table 1.

Table 1. One-photon photo-physical properties of Tp-bis(StACN-TPA).

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An example UV-Vis and PL spectrum in THF of a 6.25 µg/mL concentrated solution is shown in Fig. 2. Tp-bis(StACN-TPA) shows three absorbance maxima in THF at 490 nm, 360 nm and 300 nm. The extinction coefficient in THF was determined as 7.2±0.1·10⁴ L·mol⁻¹ · cm⁻¹ using 9 samples with concentrations from 0.05 mg/mL to 1.95 · 10⁻⁴ mg/L. For other solvents only one concentration was used and therefore these values are afflicted with a higher error. Due to the limited solubility of Tp-bis(StACN-TPA) in 2-methyl-2-butanol the calculated values need to be considered with caution [9]. For all PL measurements excitation was performed at 490 nm. Quantum yields were determined by using Rhodamin 6G as a reference. The maximum of the PL intensity can be seen at 678 nm for the solution in THF. Fluorescence quenching occurs at concentrations of 12.5 µg/mL (1.43 µmol/L) or higher.

 

Fig. 2 (a): UV-Vis and PL spectra of Tp-bis(StACN-TPA) (c = 6.25 µg/mL). (b): Integrated PL intensity versus concentration of Tp-bis(StACN-TPA) in THF.

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There is only a small spectral overlap within the UV-Vis and PL spectra, therefore the chance of quenching due to Förster resonance energy transfer is low [3]. The calculated and observed absorption maxima of Tp-bis(StACN-TPA) (see Table 2 in section 3.3) are closer to each other compared to the calculated and observed emission maxima. In solid state high fluorescence intensity can be observed which can be explained by stacking-induced planarization. This might diminish the non-radiative decay pathway due to molecular movements. Furthermore, it is proposed that even in solid state a certain amount of distortion is maintained. This explains the high PL intensity in solid state as usually intermolecular quenching effects occur in close packings [4].

Table 2. Calculated optical properties of Tp-bis(StACN-TPA).

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Solvatochromic effects were studied using the Lippert-Mataga model as shown in Fig. 3. The solvent polarities were calculated using the Lippert-Mataga equation Eq. (1) with n as the refractive index and ε as the dielectric constant [3, 10].

No solvatochromism is noticeable in the absorbance data, therefore it seems that the ground state, which usually has a lower dipole moment compared to the excited state, is scarcely affected by solvent polarity in the case of Tp-bis(StACN-TPA). However, the emission spectra are strongly influenced by the solvent polarity. In nonpolar solvents like toluene a high quantum yield of 49 % and an emission maximum at 591 nm are observable. With increasing solvent polarity the emission maximum is red-shifted and the quantum yield decreases linearly Fig. 3(a).

 

Fig. 3 (a):Dependence of the quantum yield of the solvent polarity and different excitation states. (b):PL spectra of Tp-bis(StACN-TPA) in different solvents. (c):Solvatochromism according to Lippert-Mataga model.

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This behavior is in line with the Lippert-Mataga model until 2-methyl-2-butanol with a solvent polarity parameter of 0.187 is used for the measurements. At this point a bimodal emission spectrum as shown in Fig. 3(b) can be measured with maxima at 632 nm and 678 nm. For solvents with higher polarity only a strong red shifted maximum is found and the quantum yield decreases below 1 %. The observed bathochromic shift exceeds the expected behavior described by the Lippert-Mataga model Fig. 3(c).

It should be noted that the Lippert equation is only an approximation for solvatochromism and the interpretation of solvent-dependent emission spectra is a very complex topic, as the emission is not only affected by solvent polarity, but also by various factors such as fluorophore-solvent and probe-probe interactions, solvent viscosity and the formation of ICT states [3].

The bimodal emission spectrum of Tp-bis(StACN-TPA) in 2-methyl-2-butanol can be interpreted as superposition of the spectra of ICT and locally excited (LE) state. The ICT state is stabilized in polar solvents and therefore the emission maximum is shifted to longer wavelengths. Furthermore, due to the strong interactions between the ICT state dipole and the solvent molecules, the lifetime of the ICT state might be increased, which could explain the low quantum yields below 1 % for Tp-bis(StACN-TPA) in solvents with high polarity [11]. In nonpolar solvents the LE state, showing no charge separation, represents the energetically lowest excited state [3]. Therefore, high quantum yields and emission at shorter wavelengths can be observed.

3.3. Quantum chemical calculations

To correlate the optical properties to the chemical structure of Tp-bis(StACN-TPA) quantum chemical calculations using DFT methods were carried out. The DFT-B3LYP hybrid functional with 6-31G** as basic set was used on the Firefly (PC GAMESS) program package to obtain an optimized structure for the ground state [12, 13]. The dihedral angle between the central acrylonitrile-thiophene group and the stilbene group is 20°. Overall, between both triphenylamine groups a twist of less then 5° was calculated.

The dihedral angle between the N-phenyl rings and the 4-styrylaniline is 80°. From the calculated data the molecular distortion is rather low, therefore extended conjugation can be expected. However, the geminal N-phenyl groups are highly twisted and are located out of the conjugation plane. These may prevent closed stacking in solid state. Tranisition energy and molecular levels were calculated using time dependend DFT with B3LYP/6-31G** as basic set. As shown in Fig. 4 the HOMO is evenly delocalized throughout the entire molecule except for the N-phenyl rings. The LUMO is localized on the core of the molecule. These orbitals show the internal charge transfer (ICT) characteristics of a D-A-D type molecule. The π-electrons are redistributed from the donor end-groups to the acceptor center.

 

Fig. 4 Illustration of molecular orbitals of Tp-bis(StACN-TPA).

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3.4. Non-linear optical properties

The two-photon cross section of Tp-bis(StACN-TPA) in THF was measured using a Ti-sapphire mode-locked femtosecond laser. As this method depends on the available laser sources, only the range from 720 nm to 890 nm was measurable with the available instruments. These wavelengths correspond to 360 nm to 445 nm of one-photon absorbance. Unfortunately these wavelengths represent a local minimum in the absorbance spectrum of Tp-bis(StACN-TPA). For the TPACS at 890 nm a value of 675±225 GM was measured.

It is well established that for centrosymmetric molecules like the one studied here, that the first two-photon allowed state is located at a higher energy than the first one-photon allowed state [1, 14]. We therefore expect this molecule to show maximum 2PA in solution state at a wavelength close to but lesser than double the one-photon maximum at 490 nm in THF. Despite this the TPACS values of 675±225 GM is on the higher side of this type of dyes [15]. Furthermore, the symmetry considerations mentioned above strongly suggest the probability of higher values of TPACS as the excitation wavelength approaches 980 nm.

3.5. Aggregation enhanced fluorescence

Aggregation enhanced fluorescence was investigated using a simple precipitation method. THF water solutions of Tp-bis(StACN-TPA) with a concentration of 6.25 µg/mL were measured regarding their absorbance and PL. As shown in Fig. 5(a), the PL intensity decreases with the addition of only a small amount of water to the THF solution.

 

Fig. 5 (a): PL intensity depending in the fraction of THF from 10 to 100 %. (b): PL spectra of pure Tp-bis(StACN-TPA) in pure THF compared to THF:water = 2:8.

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A local minimum at a fraction of 40 % water can be observed, followed by increasing PL intensities with increasing amount of water. At a fraction of 80 % water the PL intensity is 200 % of the initial value of a THF solution. This effect can be correlated with the observed solvatochromism behavior. Low fractions of water seem to increase the solvent polarity and therefore decrease the quantum yield by favoring the ICT state, while the solubility of the dye is still sufficient. At about 40–60 % fraction of water the formation of bulk precipitates visible by eye occurs slowly within 10 to 20 minutes after preparation of the samples. The measured PL intensities of these THF water mixtures are low at the given sample concentration of 6.25 µg/mL. This might be due to the low surface to volume ratio of the bulk precipitates. With further water addition the solubility of Tp-bis(StACN-TPA) in the THF water mixtures is further reduced and therefore the formation of aggregates occurs much faster. Similar behavior was reported by Prasad et al. [4]. However, it should be noticed that a higher concentrated sample (c=0.05 mg/ml) shows 5 times the PL intensity compared to a THF solution when bulk precipitation occurs at a water fraction of 50 %. This increase can be explained as the THF solutions show intense quenching effects at concentrations higher than 12.5 µg/mL. This relativises the seen effect. At a fraction of 70 to 90 % water light scattering can be observed due to the formation of nanoaggregates. These small aggregates show a locally nonpolar environment that favors the LE state, hence increasing the PL intensity while showing a high surface to volume ratio. Moreover, a behavior similar to solid state can be expected. As shown in Fig. 5(b) a blue shift of the PL maximum compared to a THF solution can be observed. Furthermore, the shoulder peak increases to form a separated peak showing the LE state. At higher amounts of water the PL intensity surprisingly decreases again. This behavior was described by Tang et al. [16].

At a fraction of 90 % water Tp-bis(StACN-TPA) seems to agglomerate too fast, thus forming amorphous particles. The aggregation enhanced fluorescence seems to be dependent on concentration and water to THF ratio and therefore might be a function of the size of the aggregates and their structure.

There are a number of well-established techniques to deliver dyes showing aggregation enhanced fluorescence into cells. A popular approach involves the use of hydrophilic micelles with or without incorporated targeting functionalities encapsulating the nanoaggregates of the dyes [17, 18]. Alternatively, dyes have been encapsulated into silica nanoparticles to deliver them into cells [19, 20]. These methods also provides the flexibility to work around the varying solubility of the dye in water. In our opinion both methods are suited for delivering Tp-bis(StACN-TPA) into cells for in vitro studies. Studies investigating Tp-bis(StACN-TPA) encapsulated in micelles are currently in progress and will be reported soon in another communication.

4. Conclusion

A new 2,5-bis(phenylacrylonitrile)thiophene based 2PA-dye has been synthesized and quantum chemical calculations have been performed. From the experimental results it has been shown that solvent polarity has a crucial influence on the excited states of Tp-bis(StACN-TPA). The formation of aggregates can be used as a viable method to overcome the low quantum yields in polar solvents. Further investigation regarding the size and structure of the aggregates needs to be done to gather insights about the parameters necessary for creating optimal conditions for the practical use of aggregation enhanced fluorescence.