Perylene diimide-based supramolecular polymer with temperature-sensitive ratiometric fluorescence responsiveness in solution and gels

The quadruple hydrogen bonding UPy units are designed to conjugate with unsubstituted PDI to exhibit bright red emission in aggregated state, together with temperature-dependent ratiometric fluorescence property in solution and hydrogels.

The integration of dyes into a supramolecular system is an efficient method to create thermo-sensitive fluorescence materials. [16][17][18] Among various supramolecular systems, supramolecular assemblies based on multiple H-bonding interactions have been proven to be a promising way to construct novel fluorescent supramolecular polymers with controllable, stimuli-responsive, and reversible features. [16][17][18][19][20][21][22][23] In these systems, fluorophore moieties are conjugated with H-bonding units to form fluorescence building blocks. [16][17][18][19][20][21][22][23] When the temperature lowers to allow the formation of directional H-bonding to order the fluorophores, the materials normally show weak fluorescence due to the quenching effect. 24 It is difficult to obtain ratiometric fluorescence, especially for the normal fluorophores that show obvious aggregation-caused quenching (ACQ) in the solid-state or high concentration of solution.
Perylene diimide (PDI) is one of the most well-known fluorescent building blocks to fabricate supramolecular fluorescence materials. 25 It has been identified as an ideal candidate for fabricating organic optoelectronic devices, dye lasers, optical switches, and fluorescent sensors. [25][26][27][28][29][30][31] And it exhibits numerous appealing features including easy functionalization, excellent stability, and ultrahigh fluorescence quantum yields in the molecular state. [32][33][34][35] In spite of these advantages, traditional PDI-based supramolecular materials usually suffer from relatively weak fluorescent intensities of redshift emission, low quantum yields, and undesirable ACQ in aggregated states due to the strong intermolecular π−π stacking of PDI plane. [36][37][38][39] Thus, the optimization of the supramolecular structure to modulate π−π stacking of PDI is essential to achieve the bright emission in the aggregated state, which can be realized by the introduction of various aromatic rings as isolation groups from the sides. 37,38 However, this strategy is often hindered by the complicated synthesis routes, and the obtained dyes generally exhibit a stationary red or deep red emission nature. 40,41 Herein we report our strategy to prepare PDI-based ratiometric fluorescence supramolecular materials that are thermo-sensitive. In our strategy, the highly directional quadruple hydrogen bonding units of ureidopyrimidinone (UPy) is designed to conjugate with PDI to control the intermolecular π−π stacking for endowing the dye with bright red emission in aggregated state, together with temperaturedependent ratiometric fluorescence property. We demonstrate this concept by comparing three rationally designed amphiphilic PDI derivatives as UPy-PDI-UPy, UPy-TPDI-UPy and TEG-PDI-TEG (Scheme 1) which are composed of a hydrophobic PDI core and two hydrophilic wings. Thanks to the unsubstituted core and linked quadruple H-bonding group (UPy) as wings, only the supramolecular polymer UPy-PDI-UPy exhibited intense fluorescence emission in the solid-state and temperature-dependent ratiometric dual-emission (green and red) property in nanoparticle dispersion when temperature elevated from 20 to 80 °C. It is worth to note that the subsequently prepared fluorescent hydrogel displayed temperature-ultrasensitive ratiometric fluorescence responsiveness as well as visual sol-gel transition ranging from 20 to 39 °C. To the best of our knowledge, this is the first example to fabricate PDI-based supramolecular polymers as ratiometric fluorescence thermometers and can imply more inspirations to prepare novel responsive luminescent materials (Scheme 1). Scheme 1. a) Molecular structure of three PDI derivatives; 2. b) Schematic illustration of temperature-sensitive supramolecular fluorescent nano-assemblies.

Materials and methods
Unless otherwise stated, all the chemicals were purchased from commercial sources and directly used in the reaction. The detailed synthesis routes of relevant intermediates such as UPy-SH, OH-PDI-OH (2a) and OH-TPDI-OH (2b), as well as the final products including UPy-TPDI-UPy and TEG-PDI-TEG (Scheme S1, S2 and S3), relevant apparatus and characterization including NMR spectra, mass spectra (MS), UV-Vis absorption and fluorescence spectra, fluorescence lifetime, atomic force microscope (AFM) and control experiments were displayed in supporting information.

Synthesis and Characterization of PDI derivatives
The chemical structures of designed PDI derivatives are shown in Scheme 1. TEG-PDI-TEG consists of a PDI core and two hydrophilic 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethanol (TEG) wings. UPy-PDI-UPy is made from a TEG-PDI-TEG structure and two ureido-4-pyrimidinone (UPy) terminal groups. Such a structure allowed UPy-PDI-UPy to form supramolecular polymers via the self-complementary quadruple H-bonding between the UPy moieties. 18,19 Compared to UPy-PDI-UPy, UPy-TPDI-UPy has a tetrachloro-substituted PDI core. The substituted groups in TPDI was designed to weaken the π−π stacking interaction of the PDI core. 42,43 These PDI derivatives were synthesized by coupling TEG/UPy with bis-N, N'-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)-3, 4, 9, 10-perylenetetra-carboxylic diimide (OH-PDI-OH) or bis-N, N'-(2-(2-(2-(2-hydroxyethoxy)ethoxy) ethoxy) ethyl)-1, 6, 7, 12tetrachlorol-3, 4, 9, 10-perylenetetracarboxylic diimide (OH-TPDI-OH). The detailed synthesis pathways are described in the supporting information. The molecular structure of three compounds and their intermediate products were confirmed by NMR spectra and mass spectra (Fig. S1-S8). The obtained three target molecules all exhibit good solubility in some organic solvents including dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), etc., and in DMF/H 2 O mixed solvent (in either molecular dispersion or aggregation state), due to the presentation of the long hydrophilic chains. Fig. 1 shows the fluorescent properties of three PDI derivatives  . 1a). Such a unique property is supported by their fluorescence spectra (Figure 1b) in which the maximum fluorescence emission intensity of UPy-PDI-UPy in solid state at 694 nm is 53 times or 41 times higher than those of TEG-PDI-TEG at 693 nm or UPy-TPDI-UPy at 636 nm. The absence of emission in both PEG-PDI-PEG and UPy-TPDI-UPy was attributed to "aggregation-caused quenching" (ACQ) effect. It is well documented that the fluorescence of PDIs is generally quenched in the solid-state due to consecutive π−π interactions and/or dipole−dipole interactions between the neighboring planar PDI fluorophores. 39 Würthner and co-workers have demonstrated that bulky substituents on PDI fluorophore favor the formation of discrete PDI−PDI π-dimers and undistorted planar PDI fluorophores, which inhibit long-range PDI aggregation in the solid state. 44,45 As displayed in the 1 H NMR spectrum in Fig. S9, with increasing the concentration of UPy-PDI-UPy, the peaks at 8.2 and 8.4 ppm belong to the PDI−PDI π-dimers gradually raise, indicating that the bulky UPy side groups in UPy-PDI-UPy can lead to the formation of discrete PDI−PDI π-dimers. 46, 47 Also, no chemical shifts relate to trimer or other long-range PDI aggregation can be observed in Fig.  S9. We thus attribute the high deep-red emission efficiency of UPy-PDI-UPy in solid state to the appropriate π−π stacking of PDI induced by UPy dimerization. Because of their amphiphilic structures, the PDI derivatives could self-assemble in aqueous solutions. UV-Vis absorption and fluorescence spectroscopies were used to study their self-assemblies (Fig. 2). A DMF/H 2 O mixture solvent was employed by increasing the fraction of water in DMF. In a concentration of 50 μM, UPy-PDI-UPy completely dissolved in pure DMF, showing typical absorption and emission images of well-dispersed PDI derivatives ( Fig. 2a and 2b). 48,49 When the content of water was increased to 30 vol%, dramatic changes in both absorption and emission spectra were observed. The S 0-1 transition (λ=490 nm) is more intense than the S 0-0 transition (λ=525 nm) while fluorescent intensity at around 545 nm is significantly reduced, indicating a π−π stacking aggregation state. With further increase of the water fraction, a slight bathochromic shift of λ max occurred and the well-structured absorption curve turned into a less structured peak. The absorbance ratio of A 525 /A 490 was declined from 1.6 to 0.4 (Fig. S10a), indicating enhanced π−π stacking interaction. 48 Interestingly, with further increasing water fraction, UPy-PDI-UPy shows a significant enhancement of fluorescent intensity at 646 nm, accompanied by a change of emission color from green to red. This unusual enhancement in emission is different from most previous reports of the PDI derivatives even UPy-linked ones. 48,49 We attributed this to the formation of appropriate intermolecular π−π stacking through multiple H-bonding interactions. Such a synergetic effect was supported by the results from UPy-TPDI-UPy in which the tetrachloro substitutes prevent effect intermolecular π−π stacking interaction and TEG-PDI-TEG, a PDI derivative without UPy moieties. UPy-TPDI-UPy only exhibits slight change in absorption along with the absorbance ratio of A 518 /A 490 (Fig. S10b), and obvious fluorescence quenching at higher water fraction (Fig. 2c and  3d). On the other hand, TEG-PDI-TEG shows obvious decrease in the absorption peak at S 0-0 transition (Fig. 2e) but stepwise promotion in that of the S 0-1 transition. As a result, the absorbance ratio of A 525 /A 490 declines from 1.45 to 0.7 (Fig. S10c). In company with this change, clear quenching in fluorescence intensity was observed, due to the formation of strong π−π interaction mediated aggregates, as similar to the literatures. To further investigate the aggregation behavior of UPy-PDI-UPy in DMF/H 2 O mixed solution, the AFM, TEM and DLS were employed to study the size of its aggregation under different water fraction. As exhibited in Fig. 3 and Fig. S11, when the water fraction was increased from 20 % to 80 %, the particle size data from DLS gradually decrease and the size distribution was also reduced. The results were also confirmed by AFM and TEM measurement. Fig. 3a and 3c displays the AFM images of the supramolecular aggregates of UPy-PDI-UPy in DMF/H 2 O mixed solution with 30 % or 50% fraction. In the case of 30 % water fraction, the aggregates show an average size of 396.5 nm (DLS). Increasing water fraction reduces both the average particle size and size distribution. The TEM images as shown in Fig. S12 further confirm the aggregation behavior of UPy-PDI-UPy in the mixture of DMF/H 2 O. This indicated the solvent polarity can be used to regulate the supramolecular selfassembly behavior of UPy-PDI-UPy in current system.

Ratiometric fluorescence responsiveness of temperature in solution and gels
Temperature-dependent fluorescence emission spectra of UPy-PDI-UPy nanoparticles dispersion in DMF/H 2 O mixed solution (v/v=50/50) were recorded from 20 °C to 80 °C (Fig. 4). With elevating the temperature, the fluorescence peak at 646 nm decreases and the emission around 545 nm becomes dominant (Fig. 4a). The average lifetime of the dispersion at 646 nm also declines from 15.9 ns to 8.9 ns (Fig. S13). The change in emission was attributed to the disassembly of PDI cores associated by both π−π stacking and Hbonding interactions. The absorption spectra of the dispersion as shown in Fig. S14 supported the conclusion that π−π stacking interaction was reduced with temperature promotion. In this process, particle size was reduced (Fig. S15), due to the segmental disaggregation of the formed supramolecular aggregates. Notably, a prominent linear fitting curve with a correlation coefficient of R 2 = 0.98, was also achieved by plotting the logarithm of I 545 /I 646 versus temperature (20.0-80.0 °C). This perfect fitting implied that the nanoparticle dispersion can be served as a favorable ratiometric fluorescent sensor for temperature. In addition, when the temperature was alternated between 20 and 80 °C, the nanoparticle dispersion exhibits the reversibly switchable fluorescence nature (Fig. 4c). A slight "fatigue" effect was observed in reversibility (Fig. 4c) and fluorescence brightness (Fig. 5d). As shown in Fig. S16, when the temperature was decreased from 80 °C to 20 °C, some big UPy-PDI-UPy nanoparticles form, which increases the distribution of nanoparticles. This result is significantly different from that of the nanoparticles formed in the mixture of DMF/H 2 O solvent ( Fig. 3c  and 3d). The different assemble strategies make the color and the fluorescence emission spectra of the nanoparticles (Fig. 4d and Fig.  S17) different from that of the initial sample at 20 °C. In addition, the "fatigue" emission still exists, even the incubation time of cooling prolong to 46 h (Fig. S17). However, marked fluorescent switching still can be visualized after five test cycles. In addition, the photostability of UPy-PDI-UPy nanoparticles in the mixture of DMF/H 2 O was provided in Fig. S18. The nanoparticles displayed good photostability under the continuous excitation of 490 nm for 60 min. Thus, the nanoparticles dispersion implied an attractive property for renewable temperature sensing in various applications. A UPy-PDI-UPy hydrogel could be prepared by adding polyethylene glycol (PEG, Mw=10,000) into the solution. This hydrogel (Gel1) was also thermo-sensitive. Elevating temperature from 20 to 39 °C induced a sol-gel transition (Fig. 5a). Interestingly, Gel1 displays obvious fluorescence color change from pink to greenyellow, accompanied by the sol-gel transition. The emission was further studied by fluorescence spectroscopy. As displayed in Figure 5b, UPy-PDI-UPy in hydrogel (Gel1) exhibits high thermosensitivity in fluorescence, even more sensitive than that in solution state. The ratiometric fluorescent intensity I 542 /I 646 was further used to evaluate the thermo-sensitivity (Fig. 5c). It was found that a sharp change in I 542 /I 646 (1.1 to 5.3) occurred in the range of 35-41 °C (critical temperature range). We attribute this to the phase-transition temperature of PEG in as-prepared hydrogel intensify the disaggregation of formed supramolecular aggregates during the phase-transition process. Moreover, a good linear fitting curve with a correlation coefficient of 0.98 (R 2 ) was achieved by plotting the I 542 /I 646 versus temperature (35.0-41.0 °C). Note that this change is perfectly matched the human physiological temperature range, implying a potential application of body-monitor. A temperaturesensitive color-changing bracelet by encapsulation of Gel1 in a plastic tube was thus fabricated to show this potential (Fig. 5a). In addition, the Gel1 also exhibits excellent reversibility in thermoinduced fluorescence change between 20 and 39 °C (Fig. 5d). In comparison with the corresponding nanoparticle dispersion, no detectable "fatigue" effects were observed in the reversibility even Please do not adjust margins Please do not adjust margins after five cycles. Furthermore, the critical temperature range of the gel can be easily varied by changing the ratio and molecular weight of PEG, as exhibited in Table S1 and Fig. S19-S21. In current system, PEG is the major component (58 and 75 wt% for Gel1 and Gel2, respectively) and water is absorbed in the PEG as a plasticizer in the gel state. PEG with M w of 10000 shows a melting point of ca. 60 °C which is expected to decrease after the addition of water. The gel-sol transition is the melting of the mixture of PEG and water. This hypothesis is also supported by the system of the PEG with lower M w (2000). Decreasing the Mw can reduce the melting point of PEG itself and also decrease the transition temperature of the system (System of Mw 10000: 41 °C; System of M w 2000: 36 °C). Based on this mechanism, the concentration of PEG should make an important contribution to the phase transition, as the results shown in Table S1 and Fig. 5a and Fig. S19 (39 °C for Gel1 and 41 °C for Gel2). In this system, the UPy-PDI-UPy molecules disperse in the media and would thus change its aggregated state with the media. Moreover, similar to UPy-PDI-UPy, TEG-PDI-TEG can also form a hydrogel. However, in contrast with the obvious temperaturedependence of UPy-PDI-UPy hydrogel, the corresponding TEG-PDI-TEG in gel (Gel4, Table S1, Fig. S19 and S22) did not show any fluorescence color change except the presence of fluorescence quenching and gel-sol transition with the increase of temperature.

Conclusions
In summary, we have synthesized three amphiphilic PDI derivatives, PEG-PDI-PEG, UPy-PDI-UPy, UPy-TPDI-UPy. They have PDI core and PEG/PEG-UPy wings. It was found that UPy-PDI-UPy exhibited intense deep-red fluorescence emission not only in solidstate, but also in the nanoparticle dispersion, implying an aggregation-induced-enhancement fluorescence. We attributed such enhancement in fluorescence to the formation of appropriate π−π stacking. Interestingly, UPy-PDI-UPy in DMF/H 2 O mixture solution (the volume ratio of H 2 O is 50%) displayed obviously ratiometric fluorescence responsiveness against temperature in the range of 20-80 °C, together with good reversible cycle property. Moreover, the as-prepared fluorescent gels consisting of UPy-PDI-UPy and PEG not only exhibited visual sol-gel transition, but also showed temperature-sensitive ratiometric dual-emission signals at a narrow range of 20-39 °C with excellent renewable nature, which is perfectly matched the human physiological temperature range. Those temperature-dependent ratiometric emission properties suggested potential application in various visual luminescent devices such as anti-counterfeiting labels and ratiometric fluorescent thermometers.

Conflicts of interest
The authors declare no competing financial interest.