Tetraphenylethene Derivatives Bearing Alkylammonium Substituents: Synthesis, Chemical Properties, and Application as BSA, Telomere DNA, and Hydroxyl Radical Sensors

Abstract

Tetraphenylethene derivatives (TPEs) are used as luminescence probes for the detection of metal ions and biomolecules. These sensors function by monitoring the increase in the photoluminescence (PL) intensity of the TPEs resulting from aggregation-induced emission (AIE) upon interaction with the analytes. The AIE behavior of the sensors was investigated by measuring their PL. In this study, PL, PL lifetime, and confocal laser scanning microscopy measurements were carried out as part of our in-depth investigation of AIE behavior of TPEs for the detection of biomolecules and radical species. We used 1,1,2,2-tetrakis(4-((trimethylammonium)alkoxy)phenyl)tetraphenylethene tetrabromide (TPE-C(m)N⁺Me₃Br⁻, m = 2, 4, and 6, where m denotes the number of methylene groups in the alkyl chain) and TPE-C(m)N⁺Me₃TCNQ^−• (TCNQ^−• is the 7,7′,8,8′-tetracyanoquinodimethane anion radical) as luminescent probes for the detection of bovine serum albumin (BSA), DNA, and the hydroxyl radical (^•OH) generated from Fenton’s reagent. The sensing performance of TPE-C(m)N⁺Me₃Br⁻ for BSA and DNA was found to depend on the length of the alkyl chains (m). UV-vis and PL measurements revealed that the responses of TPE-C(m)N⁺Me₃Br⁻ and TPE-C(4)N⁺TCNQ^−• to Fenton’s reagent depended on the solvent. The electrochemical properties of the TPE derivatives prepared in this study were additionally investigated via cyclic voltammetry.

1. Introduction

The aggregation-induced emission (AIE) of tetraphenylethene derivatives (TPEs) is the result of the inhibition of bond rotation between the phenyl and ethene groups when these derivatives form aggregates [1,2,3]. TPE derivatives are used as luminescence probes for the detection of metal ions as well as biomolecules such as bovine serum albumin (BSA) and DNA [4,5,6,7,8]. These sensors operate by monitoring the AIE behavior attributable to the interaction between the TPE derivatives and analytes.

BSA has been extensively studied owing to its structural homology with human serum albumin. Tang reported that an anionic TPE derivative, 1,2-bis(4-(3-sulfonatoproxyl)phenyl)-1,2-diphenylethene sodium salt (1) [9], and a cationic TPE derivative, 1,2-bis[(4-((trimethylammonium)ethyl)phenyl)-1,2-diphenylethene dibromide (2) [10], could detect BSA by monitoring the increase in the photoluminescence (PL) intensity upon the addition of BSA to solutions of these TPE derivatives. Specifically, the anionic and cationic TPE derivatives interact electrostatically with the ammonium cation and carboxyl anion of BSA, respectively. The increase in the PL intensity of 1 and 2 upon the addition of BSA is the result of AIE. In this study, 1,1,2,2-tetrakis(4-((trimethylammonium)alkoxy)phenyl)tetraphenylethene tetrabromide (TPE-C(m)N⁺Me₃Br⁻; m = 2, 4, and 6, where m denotes the number of methylene groups in the alkyl chain) was used for the detection of BSA. TPE-C(m)N⁺Me₃Br⁻ is expected to interact electrostatically with BSA via its four ammonium groups, thereby inhibiting the rotation of all of the bonds between the ethene and phenyl groups. This inhibition is anticipated to give rise to effective AIE and enable analytes to be detected with high sensitivity. Because the PL lifetimes of π-conjugated molecules have been reported to increase when they aggregate in solution [11,12], PL lifetime (t’s) measurements and confocal laser scanning microscopy (CLSM) analyses of the TPE-C(m)N⁺Me₃Br⁻ solutions were conducted before and after the addition of BSA. This information was expected to advance our understanding of the AIE behavior of the TPE derivatives when they are used as sensing probes.

In addition to the detection of BSA, we investigated the telomere DNA sensing behavior of TPE-C(m)N⁺Me₃Br⁻. Telomere DNA is an essential part of human chromosomes that regulates cell ageing. Human telomere DNA is composed of a repeating sequence of nucleotides, namely TTAGGG (T = thymine, A = adenine, and G = guanine). Guo reported that 1,1,2,2-tetrakis(4-((trimethylammonium)butyl)phenyl)tetraphenylethene tetrabromide (TPE-C(4)N⁺Me₃Br⁻) could detect the chain lengths of GGG(TTAGGG)_n (n = 3, 7, 8, 9, 10, and 11). This sensor is based on the occurrence of AIE upon the addition of DNA to the TPE-C(4)N⁺Me₃Br⁻ solution [13,14]. However, with the exception of TPE-C(4)N⁺Me₃Br⁻, TPE-C(m)N⁺Me₃Br⁻ has not been used for DNA sensing. The length of the alkyl chain (m) of TPE-C(m)N⁺Me₃Br⁻ is likely to affect the interaction between the ammonium group of TPE-C(m)N⁺Me₃Br⁻ and the phosphate group of DNA. With this in mind, we investigated the extent to which the length of the alkyl chain of TPE-C(m)N⁺Me₃Br⁻ affects the PL intensity and lifetime by measuring these quantities before and after the addition of (TTAGGG)_n (n = 2, 4, and 6). These data are useful for understanding the AIE behavior of TPE derivatives with respect to DNA sensing.

Alternative applications of TPE-C(m)N⁺Me₃Br⁻ (m = 2, 4, and 6) were explored by studying the anion exchange reaction of TPE-C(m)N⁺Me₃Br⁻ with the lithium salt of the 7,7′,8,8′-tetracyanoquinodimethane anion radical (TCNQ^−•). To the best of our knowledge, the products of this reaction, TPE-C(m)N⁺Me₃TCNQ^−•, are the first examples of TPE derivatives bearing TCNQ^−•. TPE-C(m)N⁺Me₃TCNQ^−• are expected to exhibit reactivity toward radical species owing to the presence of TCNQ^−•. We previously reported that the TCNQ^−• group in cationic polymers acted as a terminator for the radical polymerization of methyl methacrylate and styrene, affording graft polymers with polymethylmethacrylate and polystyrene chains bonded to TCNQ⁻ [15].

The detection of the hydroxyl radical (^•OH) has attracted attention because it is known to inflict damage upon biomolecules such as amino acids, lipids, and nucleic acids [16]. Several methods to detect ^•OH with the help of fluorescent probes, such as coumarin derivatives [17,18,19,20,21], fluorescein derivatives [22], and fluorescent aromatic compounds with the 2,2,6,6-tetramethylpiperidine-1-oxyl moiety, have been reported [23]. Depending on the solvent, radical species such as ^•OH and ^•CH₃ can be generated with the aid of Fenton’s reagent [24]. The detection of biomolecules and ^•OH should preferably be conducted in water. However, the probes that were initially reported were used in organic solvents and mixtures consisting of water and an organic solvent because they were insoluble in water. In contrast, TPE-C(m)N⁺Me₃Br⁻ can be used as sensing probes in both water and organic solvents because of their high solubilities. In this study, we investigated the PL and UV-vis responses of TPE-C(m)N⁺Me₃Br⁻ and TPE-C(4)N⁺Me₃TCNQ^−• to Fenton’s reagent. To the best of our knowledge, the use of TPE derivatives for the detection of radical species has not yet been reported. This work is expected to unveil fundamental information on the development of new probes based on TPE derivatives for the detection of radical species.

Herein, we report the synthesis, and the optical, and electrochemical properties of TPE-C(m)Br, TPE-C(m)N⁺Me₃Br⁻, and TPE-C(m)N⁺Me₃TCNQ^−• and their sensing performance of BSA, DNA, and radical species.

2. Results and Discussion

2.1. Synthesis

The reaction of TPE-C(m)N⁺Me₃Br⁻ (m = 2, 4, and 6) with the lithium salt of the 7,7′,8,8′-tetracyanoquinodimethane anion radical (Li⁺TCNQ^−•) proceeds via anion exchange between Br⁻ and TCNQ^−• to yield TPE-C(m)N⁺Me₃TCNQ^−• (Scheme 1). Elemental analysis indicated that this anion exchange reaction proceeded to completion. TPE-C(m)N⁺Me₃Br⁻ was soluble in water and polar organic solvents such as acetone, acetonitrile, and dimethyl sulfoxide (DMSO) but insoluble in tetrahydrofuran (THF), whereas TPE-C(m)N⁺Me₃TCNQ^−• was soluble in the polar organic solvents but insoluble in water and THF. The IR absorption band corresponding to n(CN) of TPE-C(4)N⁺Me₃TCNQ^−• was observed at 2178 cm⁻¹ (Figure S1). This wavenumber is comparable to that of Et₄N⁺TCNQ^−• (n = 2182 cm⁻¹ [25]). The chemical shifts of the ¹H NMR peaks corresponding to the TPE and alkyl protons of TPE-C(m)N⁺Me₃TCNQ^−• were almost the same as those of TPE-C(m)N⁺Me₃Br⁻. However, the ¹H NMR signal corresponding to the TCNQ^−• protons of TPE-C(m)N⁺Me₃TCNQ^−• disappeared due to the presence of the anion radical (Figure S2).

2.2. Optical Properties

Figure 1a shows the UV-vis spectra of TPE-C(4)N⁺Me₃Br⁻ and TPE-C(4)N⁺Me₃TCNQ^−• in DMSO. The UV-vis spectrum of TPE-C(4)Br closely resembles that of TPE-C(4)N⁺Me₃Br⁻. The absorptions corresponding to the π−π* transitions of the phenyl rings and the TPE core of TPE-C(4)N⁺Me₃Br⁻ were observed near 270 and 320 nm, respectively. In addition to these absorptions, TPE-C(4)N⁺Me₃TCNQ^−• exhibited absorptions corresponding to the D₀ → D₂ and D₀ → D₁ transitions of the TCNQ^−• group in the ranges of 400−480 nm and 680−850 nm, respectively. These absorption ranges are similar to those of Et₃NH⁺TCNQ^−• [26]. It appears that the alkyl chain of TPE-C(m)N⁺Me₃Br⁻ and TPE-C(m)N⁺Me₃TCNQ^−• hardly affects their optical properties as the UV-vis spectra of TPE-C(m)N⁺Me₃Br⁻ and TPE-C(m)N⁺Me₃TCNQ^−• are similar irrespective of the alkyl chain length (m), as shown in Figure S3. Figure 1b shows the photoluminescence (PL) spectra of TPE-C(4)N⁺Me₃Br⁻ in various organic solvents. The PL peak positions shifted to longer wavelengths as the donor number (DN) of the solvents increased (Figure 1c). The results indicate that the geometry of the excited state could be different from that of the ground state. The bathochromic shift of the PL peak of TPE-C(4)-N⁺Me₃Br⁻ appeared to be due to its increased stability in the excited state relative to that in the ground state. Furthermore, a large amount of stabilization energy produced via the solvation of TPE-C(4)-N⁺Me₃Br⁻ may contribute to the bathochromic shift as the DN of the solvents increases. Figure 1d shows the PL spectra of TPE-C(4)N⁺Me₃Br⁻ in DMSO and DMSO/THF mixtures. The PL intensity of TPE-C(4)N⁺Me₃Br⁻ increases as the THF content of DMSO/THF increases. The increase in the PL intensity corresponds to the AIE, attributed to the immiscibility of TPE-C(4)N⁺Me₃Br⁻ with THF. The confirmation that TPE-C(4)N⁺Me₃Br⁻ underwent AIE was evident from the longer PL lifetimes (t’s) of TPE-C(4)N⁺Me₃Br⁻ with an increase in the THF content of DMSO/THF (Figure 1f). The fluorescence decay profiles are shown in Figure 1e. As mentioned above, the PL lifetime (t) of π-conjugated molecules increases with aggregation [11,12]. Contrary to TPE-C(4)N⁺Me₃Br⁻, TPE-C(4)N⁺Me₃TCNQ^−• dissolved in DMSO did not undergo AIE in DMSO/THF. This result corresponds to the assumption that bulky TCNQ^−• groups prevent the aggregation of TPE-C(4)N⁺Me₃TCNQ^−• in solutions.

2.3. Electrochemical Properties

The electrochemical properties of TPE-C(m)Br, TPE-C(m)N⁺Me₃Br⁻, and TPE-C(m)N⁺Me₃TCNQ^−• were investigated via cyclic voltammetry (CV). Figure 2a shows the CV curves of TPE-C(4)Br, TPE-C(4)N⁺Me₃Br⁻, and TPE-C(4)N⁺Me₃TCNQ^−• in acetonitrile containing 0.10 M [Et₄N]BF₄. The TPE core of TPE-C(4)Br underwent electrochemical oxidation at E_pa(1) = 0.54 V (vs. Ag⁺/Ag). The CV curve of TPE-C(4)N⁺Me₃Br⁻ shows two pairs of peaks, which correspond to the redox reaction between Br⁻ and Br₃⁻ at E_pa(2) = 0.31 V (vs. Ag⁺/Ag), and between Br₃⁻ and Br₂ at E_pa(3) = 0.62 V (vs. Ag⁺/Ag). In addition, a shoulder peak corresponding to the electrochemical oxidation of the TPE core at E_pa(1) = 0.55 V (vs. Ag⁺/Ag) is also observed. The E_pa(2) and E_pa(3) values of TPE-C(m)N⁺Me₃Br⁻ (m = 2 and 6) were 0.31 V and 0.66 V (vs. Ag⁺/Ag) and 0.31 V and 0.62 V (vs. Ag⁺/Ag), respectively. The electrochemical reactions of the TPE derivatives are shown in Scheme 2. Reportedly, the two-step electrochemical oxidation of Br⁻ of tetraethylammonium bromide yields Br₂ in solution [27]. Figure S3 shows plots of the peak currents of the oxidation of Br⁻ to Br₃⁻ (i_pa(2)) and reduction of Br₃⁻ to Br⁻ (i_pc(2)) vs. v^1/2. Plotting the i_pa(2) and i_pc(2) values as a function of v^1/2 revealed a linear relationship, indicating that the redox reaction is diffusion-controlled [28]. The TCNQ^−• group of TPE-C(4)N⁺Me₃TCNQ^−• undergoes a two-step electrochemical redox reaction (Scheme 2d,e). The CV curves of TPEC(m)N⁺Me₃TCNQ^−• (m = 2 and 6) are shown in Figure S5. The electrochemical reduction potentials of the TCNQ^−• group in TPE-C(4)N⁺Me₃TCNQ^−• are E_pc(4) = –0.19 V and E_pc(5) = –0.74 V (vs. Ag⁺/Ag). The E_pc(4) and E_pc(5) values of TPEC(m)N⁺Me₃TCNQ^−• (m = 2 and 6) were E_pc(4) = –0.20 V and E_pc(5) = –0.74 V (vs. Ag⁺/Ag) and E_pc(4) = –0.18 V and E_pc(5) = –0.75 V (vs. Ag⁺/Ag), respectively. These values are lower than the corresponding E_pc values of TCNQ (E_pc = –0.094 and –0.64 V (vs. Ag⁺/Ag) [29]) in a solution of tetrabutylammonium perchlorate in acetonitrile. This result corresponds to the fact that the TCNQ^−• moiety in TPE-C(m)N⁺Me₃TCNQ^−• is electrostatically bonded to the ammonium group.

2.4. BSA Sensing

Figure 3a depicts the changes in the PL spectra observed for TPE-C(4)N⁺Me₃Br⁻ upon the addition of BSA in 0.1 equimolar amounts. The PL intensity of TPE-C(4)N⁺Me₃Br⁻ increased with increasing amounts of BSA. The increase in the PL intensity upon the addition of BSA was also observed for TPE-C(m)N⁺Me₃Br⁻ (m = 2 and 6) (Figure S4). Figure 3b shows the BSA concentration vs. the I/I₀ values of TPE-C(m)N⁺Me₃Br⁻, where I₀ and I denote the PL intensity of TPE-C(m)N⁺Me₃Br⁻ in the absence and presence of BSA, respectively.

These results show that TPE-C(m)N⁺Me₃Br⁻ is able to successfully detect BSA in the concentration range of 0–4 × 10⁻⁵ M. This detection range is wider than that of 2 (0–8 × 10⁻⁶ M) [10]. The extent to which the PL increased in response to the addition of BSA increased with increasing alkyl chain length (m) of TPE-C(m)N⁺Me₃Br⁻, as shown in Figure 3b. This result corresponds to the assumption that a long alkyl chain can prevent steric repulsion between the TPE core and BSA when the cationic group of TPE-C(m)N⁺Me₃Br⁻ interacts with the anionic group of BSA. The images of the interactions between TPE-C(m)N⁺Me₃Br⁻ (m = 2 and 6) and BSA or (GGGATT)_n are shown in Figure S7. The PL lifetime and confocal laser scanning microscopy (CLSM) measurements revealed that the increase in the PL intensity of TPE-C(m)N⁺Me₃Br⁻ upon the addition of BSA was attributed to AIE. The fluorescence decay profiles are shown in Figure 3c. Figure 3d shows the changes in the t values of an aqueous solution of TPE-C(m)N⁺Me₃Br⁻ upon the addition of BSA. The t values of the aqueous solution of TPE-C(m)N⁺Me₃Br⁻ increased as the BSA content of the solution increased. This result indicates that TPE-C(m)N⁺Me₃Br⁻ underwent aggregation when interacting with BSA. The samples for the CLSM measurements were prepared by casting aqueous solutions of TPE-C(4)N⁺Me₃Br⁻ on the glass plates in the absence and presence of BSA. Figure 3e,f depicts CLSM images of the TPE-C(4)N⁺Me₃Br⁻ powder (sample A) and TPE-C(4)N⁺Me₃Br⁻ cast in a BSA film (sample B) on glass plates. The CLSM image of sample A shows low-intensity bright spots, whereas that of sample B exhibits intense bright bands, indicating that the AIE of TPE-C(4)N⁺Me₃Br⁻ was the result of the interaction with BSA.

The conformations of BSA in aqueous solutions of TPE-C(m)N⁺Me₃Br⁻ were investigated by measuring the viscosity. The conformation of a macromolecule in solution is related to the a exponent of the Mark–Houwink–Sakurada equation, [η] = KM^a and the values of polymers in the expanded state are larger than those in contracted or intertwined states. Moreover, the viscosity of BSA reportedly depends on its conformation in aqueous solutions [30]. The specific viscosities (η_sp′s) of aqueous solutions of BSA (c = 2.0 g·dL⁻¹) at pH 2.7, 7.4, and 10.0 are 0.16, 0.056, and 0.058 dL·g⁻¹, respectively [30]. These data correspond to the fact that BSA has an expanded conformation in acidic solutions and a contracted conformation in neutral and basic solutions. The η_sp values of aqueous solutions of BSA (c = 2.0 g·dL⁻¹) in the presence of equimolar amounts of TPE-C(m)N⁺Me₃Br⁻ (m = 2, 4, and 6) were 0.083, 0.082, and 0.088 dL·g⁻¹, respectively. These values are smaller than the η_sp value of the aqueous solution of BSA in the absence of TPE-C(m)N⁺Me₃Br⁻ (h_sp = 0.10 dL·g⁻¹). This result indicates that the conformation of BSA forms is slightly contracted when it interacts with TPE-C(m)N⁺Me₃Br⁻.

2.5. DNA Sensing

Figure 4a depicts the changes in the PL spectra of TPE-C(4)N⁺Me₃Br⁻ upon the addition of (TTAGGG)₆ in 0.1 equimolar amounts. The PL intensity of TPE-C(4)N⁺Me₃Br⁻ increased until a 0.6 equimolar amount of DNA had been added and remained unchanged with the further addition of DNA. The PL intensity of TPE-C(m)N⁺Me₃Br⁻ (m = 2 and 4) increased upon the addition of (TTAGGG)₆ (Figure S5). These results indicate that TPE-C(m)N⁺Me₃Br⁻ can detect DNA chain lengths in the six-base order, within several minutes, confirming that TPE-C(m)N⁺Me₃Br⁻ significantly outperforms previously reported sensors. The DNA concentration vs. I/I₀ of TPE-C(m)N⁺Me₃Br⁻ is plotted in Figure 4b. The I/I₀ values of TPE-C(m)N⁺Me₃Br⁻ increased with increasing alkyl chain length (m). Figure 4c shows the (TTAGGG)₆ concentration vs. I/I₀ of TPE-C(m)N⁺Me₃Br⁻. The I/I₀ values of TPE-C(m)N⁺Me₃Br^- upon the addition of DNA increased with increasing alkyl chain length (m). The results are comparable to the PL spectral changes of TPE-C(m)N⁺Me₃Br⁻ with the addition of BSA. The I/I₀ values of TPE-C(m)N⁺Me₃Br⁻ (m = 2 and 4) increase in proportion to the amounts of DNA, whereas the I/I₀ values of TPE-C(6)N⁺Me₃Br⁻ increase until 0.5 equimolar amounts of DNA have been added, after which the values remain constant (Figure 5b). These results correspond to the assumption that TPE-C(m)N⁺Me₃Br⁻ (m = 2 and 4) and TPE-C(6)N⁺Me₃Br⁻ form complexes with DNA in 1:1 and 1:2 molar ratios, respectively. This assumption was confirmed via a Benesi–Hildebrand analysis using the following equations [31]:

1/(F₀ − F) = 1/(F₀ − F_∞)K[DNA] + 1/(F₀ − F_∞)

1/(F₀ − F) = 1/(F₀ − F_∞)K[DNA]² + 1/(F₀ − F_∞)

where F₀ and F denote the PL intensities of TPE-C(m)N⁺Me₃Br⁻ in the absence and presence of DNA, respectively, and F_∞ is the PL intensity at saturation. As shown in Figure 4d,e, straight lines were obtained by plotting the 1/(F₀ − F) values as a function of 1/[(TTAGGG)₂] in 1:1 Benesi–Hildebrand plots for TPE-C(m)N⁺Me₃Br⁻ (m = 2 and 4), and by plotting the 1/(F₀ − F) values as a function of 1/[(TTAGGG)₂]² in 1:2 Benesi–Hildebrand plots for TPE-C(6)N⁺Me₃Br⁻. In contrast, plots of 1/(F₀ − F) vs. 1/[(TTAGGG)₂]² in 1:2 Benesi–Hildebrand plots for TPE-C(m)N⁺Me₃Br⁻ (m = 2 and 4), and of 1/(F₀ − F) vs. 1/[(TTAGGG)₂] in 1:1 Benesi–Hildebrand plots for TPE-C(6)N⁺Me₃Br⁻ revealed that the relationship was nonlinear. Figure 4f shows the changes in the t values of aqueous solutions of TPE-C(m)N⁺Me₃Br⁻ upon the addition of (TTAGGG)_n (n = 2, 4, and 6). The PL lifetimes of TPE-C(m)N⁺Me₃Br⁻ were lengthened after the addition of DNA. These results confirm that the increase in the PL of TPE-C(m)N⁺Me₃Br⁻ upon the addition of DNA corresponds to AIE.

2.6. Hydroxyl Radical Sensing

Figure 5a,b shows the changes in the UV-vis spectra of TPE-C(4)N⁺Me₃Br⁻ with the addition of Fenton’s reagent in water and DMSO, respectively. The absorption at approximately 270 nm corresponding to π–π* transitions of the phenyl rings of TPE-C(4)N⁺Me₃Br⁻ increased with increasing amounts of Fenton’s reagent. However, the absorption at approximately 320 nm gradually decreased until four equimolar amounts of Fenton’s reagent had been added and disappeared with the addition of further amounts of Fenton’s reagent.

This result corresponds to the assumption that the reaction of radical species generated from Fenton’s reagent with the ethene group of TPE-C(4)N⁺Me₃Br⁻ shortens the length of the π-conjugation. In this regard, it has been reported that ^•OH reacts with unsaturated bonds such as C=C, C=O, and C=S [32]. The PL response of TPE-C(4)N⁺Me₃Br⁻ to Fenton’s reagent was solvent-dependent. Figure 5c shows the changes in the PL spectra of aqueous solutions of TPE-C(4)N⁺Me₃Br⁻ with the addition of Fenton’s reagent. The computational calculations revealed that the PL peaks of TPE at 459 nm and 540 nm corresponded to the S₃ → S₀ and S₁ → S₀ transitions, respectively [33]. As shown in Figure 5c, the peak at approximately 470 nm, which corresponds to S₁ → S₀ transition, disappeared with the addition of four equimolar amounts of Fenton’s reagent. This result is in agreement with the result that the absorption corresponding to the π–π* transitions of TPE-C(4)N⁺Me₃Br⁻ disappeared after the addition of four equimolar amounts of Fenton’s reagent. The fact that the addition of further amounts of Fenton’s reagent caused the PL peak at 410 nm, which corresponds to the S₃ → S₀ transition, to intensify was possibly attributable to the occurrence of AIE. In contrast, the PL intensity of DMSO solutions of TPE-C(4)N⁺Me₃Br^- decreased with increasing amounts of Fenton’s reagent (Figure 5d). Considering reports that ^•OH reacts with DMSO to yield ^•CH₃, the reason for the decrease in the PL in the DMSO solution of TPE-C(4)N⁺Me₃Br^- upon the addition of Fenton’s reagent is unclear. However, the geometry of the excited state of the reaction product of TPE-C(4)N⁺Me₃Br⁻ and ^•OH could differ from that of the product of TPE-C(4)N⁺Me₃Br⁻ and ^•CH₃. The extent to which the PL of TPE-C(4)N⁺Me₃Br⁻ was quenched using Fenton’s reagent was quantified using the Stern–Volmer equation, I₀/I = 1 + K_SV[quencher], where I₀ and I denote the PL intensities with and without the quencher, respectively. The Stern–Volmer plots for the PL quenching of TPE-C(4)N⁺Me₃Br⁻, shown in Figure 5e, are convex downward. This result indicates that the PL quenching is attributed to the presence of the combined effect of static and dynamic mechanisms. The occurrence of both static and dynamic quenching for the same fluorophore is represented by a nonlinear equation in terms of the quencher concentration ([Q]): F₀/F = (1 + K_D[Q])(1 + K_S[Q]), where K_D and K_S are static and dynamic Stern–Volmer constants, respectively [34]. The modified Stern–Volmer equation is a quadratic function of [Q], which accounts for the downward curvature that was observed.

Figure 5f shows the changes in the UV-vis spectra of TPE-C(4)N⁺Me₃TCNQ^−• in DMSO with the addition of Fenton’s reagent. The absorption corresponding to the D₀ → D₂ and D₀ → D₁ transitions of TCNQ^−• of TPE-C(4)N⁺Me₃TCNQ^−• decreased with increasing amounts of Fenton’s reagent. These results indicate that TPE-C(4)N⁺Me₃TCNQ^−• could be used for the detection of ^•CH₃ by monitoring the absorption changes. The disappearance of the peak corresponding to the TCNQ^−• group indicates that TCNQ^−• had reacted with ^•CH₃. This assumption is confirmed by the appearance of the peak corresponding to the TCNQ⁻ group at 329 nm. The peak appearance was accompanied by an increase in the absorption at 488 nm probably because of the formation of TCNQ²⁻. TCNQ²⁻, which is known to form via the disproportionation reaction of TCNQ⁻, exhibited an absorption maximum at approximately 500 nm [35]. The UV-vis response of TPE-C(m)-N⁺Me₃TCNQ^−• (m = 2 and 6) to Fenton’s reagent is expected to be similar to that of TPE-C(4)-N⁺Me₃TCNQ^−• as the UV-vis spectra of TPE-C(m)-N⁺Me₃TCNQ^−• (m = 2, 4, and 6) are similar irrespective of the chain length, as shown in Figure S3b. The PL response of TPE-C(4)N⁺Me₃TCNQ^−• to Fenton’s reagent in DMSO was considerably different from that of TPE-C(4)N⁺Me₃Br⁻. The decrease in the PL intensity of TPE-C(4)N⁺Me₃TCNQ^−• with the addition of Fenton’s reagent was considerably less pronounced than that of TPE-C(4)N⁺Me₃Br⁻ (Figure S6). As mentioned above, the TCNQ^−• group of TPE-C(4)N⁺Me₃TCNQ^−• reacted with ^•CH₃. However, the bulky TCNQ group may hinder the reaction of the TPE core with ^•CH₃, as is evident from the negligible changes in the PL spectra recorded after the addition of Fenton’s reagent.

3. Experimental Section

3.1. General

TPE-C(m)Br, TPE-C(m)N⁺Me₃Br⁻ (m = 2, 4, and 6) [36], and Li⁺TCNQ^−• [26] were prepared according to the literature. (TTAGGG)_n (n = 2, 4, and 6; purified via HPLC) was purchased from Nippon Gene Co. LTD. (Tokyo, Japan). Other reagents were purchased and used without further purification. Acetonitrile, dimethyl sulfoxide (DMSO), methanol, and tetrahydrofuran (THF) were obtained from WAKO Chemical Co. Limited. (Odawara, Japan). These solvents were dried, distilled, and stored under N₂. Bovine serum albumin (BSA), hydrogen peroxide, and iron(II) sulfate heptahydrate were purchased from WAKO Chemical Co. Limited. (Odawara, Japan). Silver nitrate and 7,7′,8,8′-tetracyanoquinodimethane (TCNQ) were purchased from TCI Co. Limited. (Taipei City, Taiwan). The reactions were carried out using standard Schlenk techniques under nitrogen.

IR and NMR measurements were caried out with a JASCO FT/IR-660 PLUS spectrophotometer with a KBr pellet and JEOL AL-400 spectrometers, respectively. Elemental analysis was conducted on a Yanagimoto MT-5 CHN corder. UV-vis and PL spectra were recorded on a JASCO V-560 spectrometer and a JASCO FP-6200, respectively. Photoluminescence lifetime measurements were conducted with a HORIBA FluoroCube Model:1000U. The CLSM images were obtained using a Leica TCS SP5. Cyclic voltammetry was performed with a Hokuto Denko HSV-110, and 1 cm × 1 cm and 1 cm × 2 cm Pt plates and Pt wire were used as working, counter, and reference electrodes, respectively. Tetraethylammonium tetrafluoroborate was used as an electrolyte. The scan speed was 50 mV·s⁻¹.

3.2. Synthesis of TPE-C(m)N⁺Me₃TCNQ^−•

TPE-C(4)Br (0.15 g, 0.12 mmol) was dissolved in 15 mL of dry MeOH at 80 °C. After Li⁺TCNQ^−• (0.16 g, 0.75 mmol) was added to the solution, the reaction solution was stirred at 25 °C for 36 h. The precipitate was collected via filtration and dried under vacuum to afford TPE-C(4)N⁺Me₃TCNQ^−• as a blue purple powder (0.16 g, 80%). ¹H NMR (400 MHz, DMSO-d₆): δ 6.86 (8H), 6.71 (8H), 3.99 (8H), 3.34 (8H), 3.05 (36H), 1.82 (8H), 1.71 (8H). Calcd. for (C₁₀₂H₁₀₀N₂₀O₄•0.6H₂O): C, 72.89; H, 6.07; N, 16.67. Found: C, 72.85; H, 5.79; N, 16.49.

TPE-C(2)N⁺Me₃TCNQ^−• and TPE-C(6)N⁺Me₃TCNQ^-. were synthesized in a similar manner. Data of TPE-C(2)N⁺Me₃TCNQ^−•: blue purple powder. Yield = 60%. ¹H NMR (400 MHz, DMSO-d₆): δ 6.90 (8H), 6.78 (8H), 4.35 (8H), 3.73 (8H), 3.15 (36H). Calcd. for (C₉₄H₉₂N₂₀O₄•0.3H₂O): C, 71.85; H, 5.94; N, 17.83. Found: C, 71.83; H, 5.09; N, 17.36. Data of TPE-C(6)N⁺Me₃TCNQ^−•: Blue purple powder. Yield = 42%. ¹H NMR (400 MHz, DMSO-d₆): δ 6.82 (8H), 6.67 (8H), 3.87 (8H), 3.26 (8H), 3.03 (36H), 1.69 (16H), 1.44 (8H), 1.33 (8H). Calcd. for (C₁₁₀H₁₁₆N₂₀O₄•H₂O): C, 73.39; H, 6.61; N, 15.56. Found: C, 73.41; H, 5.96; N, 14.84.

4. Conclusions

In conclusion, the chemical properties and applicability as biomolecule sensors of the TPE derivatives bearing alkylammonium bromide (TPE-C(m)N⁺Me₃Br⁻) and the alkylammonium TCNQ radical anion (TPE-C(m)N⁺Me₃TCNQ^−•) were investigated. PL measurements revealed that TPE-C(m)N⁺Me₃Br⁻ aggregated in DMSO/THF mixtures, and their PL intensities increased as the THF content increased. The CV measurements indicated that the TPE derivatives obtained in this study were electrochemically active. TPE-C(m)N⁺Me₃Br⁻ could be used as sensors for BSA and telomere DNA by detecting the changes in their PL intensities upon the addition of the analytes. The sensing performance of TPE-C(m)N⁺Me₃Br⁻ depended on the length of the alkyl chain of TPE-C(m)N⁺Me₃Br⁻. Anionic contaminants in sample solutions may affect the sensing performance of TPE-C(m)-N⁺Me₃Br⁻ because the sensors are based on the occurrence of AIE, attributed to the interaction between the cationic group of TPE-C(m)N⁺Me₃Br⁻ and the anionic group of BSA and DNA. In the future, we will study the analyte selectivity of the sensors via an investigation of the optical responses of TPE-C(m)-N⁺Me₃Br⁻ to the addition of BSA or DNA in the presence of various anionic species. TPE-C(m)N⁺Me₃Br^- and TPE-C(4)N⁺Me₃TCNQ^−• were also found to be suitable for use as sensors for radical species, as the changes in the intensity of the UV-vis and PL peaks were dependent on the amount of Fenton’s reagent. We can conclude that the results of this study indicated that TPE derivatives bearing alkylammonium salts can be used as sensing materials for biological and radical species.