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10.1021/acsomega.4c03137
Article
Series of Fluorescent Dyes Derived from Triphenylamine Structure for Cu2+ and In-Cell Carbon Monoxide Sensing: Synthesis and Performance
Fei Yulang †‡
Liu Han §
https://orcid.org/0009-0004-7138-781X
Sun Kai *‡
https://orcid.org/0009-0006-5769-4287
Zhang Ou *‡
† Department of Biomedical Research Center, Medical College, Xijing University, Xi ’an 710123, Shaanxi Province, China
‡ The First Affiliated Hospital of Nanyang Medical College, Nanyang 473061, Henan Province, China
§ Department of the Spleen and Stomach Diseases, Xi’an Hospital of Traditional Chinese Medicine, Xi ’an 710000, Shaanxi Province, China
* Email: sunkai796@163.com.
* Email: zhangou361@163.com.
26 08 2024
10 09 2024
9 36 3773737747
01 04 2024
21 08 2024
20 07 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

In this paper, triphenylamine served as the structural core and was bonded to aromatic groups having various substituents [—OH, —OMe, or —N(Et)2] by a =N—N= chain and then connected with aromatic groups having various substituents [—OH, —OMe, or —N(Et)2]. The geometric and electronic properties of these probes were examined. It was found that the presence of electron donors enhanced the selectivity and emission quantum yield (QY). When exposed to Cu2+, the fluorescence intensity decreased. The optimal probe (T5) showed a significant decrease in emission QY from 17.1 to 0.5% and recovered to 16.8% after exposure to CO for 342 s. The sensing mechanism was revealed to be static quenching, forming a nonfluorescent adduct between probe and Cu2+. After reacting with CO, Cu2+ was reduced to Cu+, and the probe emission was recovered. The bioimaging performance of the optimal probe was assessed as well.

Health Commission of Henan Province 10.13039/100018925 SBGJ202303051 Natural Science Basic Research Program of Shaanxi Province 10.13039/501100017596 2021JQ-881 Nanyang Science and Technology Information Network 10.13039/501100011495 JCQY021 document-id-old-9ao4c03137
document-id-new-14ao4c03137
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pmc1 Introduction

As the third most abundant metal ion in the human body, the divalent copper ion (Cu2+) plays a key role in various biological reactions, such as serving as a catalytic cofactor of redox-regulating enzymes such as tyrosinase, lysyl oxidase, cytochrome c oxidase, and superoxide dismutase.1 Both a lack of Cu2+ and long-term exposure to high concentrations of Cu2+ lead to health problems or diseases.2−8 For example, an accumulation of Cu2+ in the human body leads to reactive oxygen species and thus efficiently damages lipids, DNA, and proteins.2,3 A high Cu2+ concentration in the human body has been associated with various health problems, including Wilson’s, Parkinson’s, Alzheimer’s, Huntington’s, and Menkes diseases, and even acute hepatic kidney failure.4−7 There are many sources of Cu2+ release into the environment, such as natural, industrial, and agricultural procedures.9 The Environmental Protection Agency (EPA) has announced an upper limit of Cu2+ in tap water as 20–30 μM and in blood serum as 100–150 μg/dL, making the detection of Cu2+ an important task in environmental protection and health care.10−13

Among the various candidates for Cu2+ detection, optical sensing, which is based on the response of optical signals (fluorescence, absorption, or emission lifetime) to analyte, seems attractive due to its advantages of fast detection, low cost, good selectivity, and easy-to-analysis.14 There have been examples of optical sensing for Cu2+. For example, Kursunlu, Oguz, Yilmaz, and co-workers from the University of Selcuk have developed a series of promising probes based on BODIPY, pillar[5]rene, and isophthalo-NBD for the detection of metal ions, including Cu2+, Sn2+, and Hg2+. Their further applications such as toxic pollutant removal and bioimaging were developed as well.15−18 Lin and his research team developed rhodamine-based probes for Cu2+.19 Solid-state sensing of Cu2+ has been reported using probe-integrated polymetric sensing materials by Srinivasan and Deivasigamani.20 Using pyrene-based fluorescent probe, Thirumalaivasan and co-workers developed an optical sensing platform for Cu2+.21 Similarly, Huang and co-workers reported a probe based on pyrene.22 Meng and co-workers developed an aldazine-based probe for the detection of Cu2+.23 In their work, it was found that the probe emission was first quenched by Cu2+, and then, in the presence of a proper reducer such as carbon monoxide (CO), Cu2+ was reduced to Cu+, with the probe emission well recovered.22 This finding actually presents a new pathway for the detection of CO. Recent literature have identified endogenic CO as a messenger in signal transduction and therapeutic effects across vital organs such as respiratory, immune, digestive, liver, and kidney systems.24−26 Consequently, the aforementioned Cu2+-probes have found another application in the fluorescence imaging of endogenic CO.22,23 Generally, to improve photon-harvesting efficiency and decrease energy-wasting structural relaxation in the excited state, the aforementioned Cu2+-probes have large conjugation planes, such as pyrene- and aldazine-derived structures.21−23 However, it is widely reported that pyrene-like molecules tend to form excimer or exciplex structures due to the π–π attraction between the coplanar conjugation planes in these probes.27 The formation of excimer or exciplex structures affects emission wavelength or emission quantum yield (QY), resulting in unexpected results and thus compromising emission-based sensing signals.

To overcome the disadvantage of pyrene-derived probes, in this work, we intend to use triphenylamine as the structural core since triphenylamine has been proven highly luminescent by various literature.28 As shown in Scheme 1, the triphenylamine group was bonded to an =N—N= chain and then connected to aromatic groups having various substituents [—OH, —OMe, or —N(Et)2]. By comparing their photoluminescence (PL) and sensing performance, the correlation between probe molecular structure and performance can be tentatively clarified. The sensing performance and the sensing mechanism of the optimal probe were reported and discussed, which allowed its further practical application in optical sensing.

Scheme 1 Synthetic Route and Working Strategy of Five Triphenylamine-Derived Dyes Tn (n = 1–5)

2 Experiment Details

2.1 General Information

Scheme 1 presents the synthetic and working strategies of the five triphenylamine-derived probes reported in this work (denoted as T1, T2, T3, T4, and T5), along with their molecular structures. The initial materials and reagents were purchased from Innochem Company in Beijing (China) and with no need for further purification directly for synthesis, including 2-hydroxybenzaldehyde (CAS 90-02-8), 2-hydroxy-1-naphthaldehyde (CAS 708-06-5), 2-hydroxy-3-methoxybenzaldehyde (CAS 148-53-8), 4-(diethylamino)-2-hydroxybenzaldehyde (CAS 17754-90-4), triphenylamine, phosphate buffered saline (PBS), CuCl, CuCl2, and tricarbonylchloro (glycinato) ruthenium(II) (CORM-3). Sample characterization was performed on a Bruker AVANCE 300 spectrometer for nuclear magnetic resonance (NMR) analysis. Single crystal and elemental analyses were performed using a Bruker SMART APEX II X-ray single-crystal diffractometer and a Carlo Erba 1106 elemental analyzer, respectively. Theoretical analysis of the probes was performed by time-dependent density functional theory (TD-DFT) method at the RB3LYP/6-31G(d) level in vacuum using Orca (version 5.04) and Firefly (version 8.2).29 The single-crystal structure of the probes (T2, T3, T4, and T5) was used as the initial geometry. Since we failed to get a T1 single crystal, we tried to simulate a T1 structure but the geometry optimization calculation failed to converge. So, the DFT calculation (frontier molecular orbital and absorption simulation) was performed only on T2, T3, T4, and T5, not on T1. The frontier molecular orbitals and the simulated absorption spectra were plotted by wxMcMolPlt (version 7.4.4, contour value = 0.03) and Multiwfn (version 3.8, first 50 singlet transitions, broadening function = Gaussian, fwhm = 0.66667 eV), respectively. X-ray photoelectron spectroscopy (XPS) and EPR (electron paramagnetic resonance) experiments were performed using an ESCALAB 250 X-ray photoelectron spectrometer and a Bruker X-band A200 spectrometer, respectively. Fluorescence imaging was carried out using an Olympus FV1200-MPE spectral confocal multiphoton microscope. Absorption and emission spectra were determined in a CH3CN/H2O (v/v = 1:1, 1 μM) solution using a Shimadzu UV-3101PC spectrophotometer and a Hitachi F-7000 fluorescence spectrophotometer (fluorescence mode, 2 × 2 nm). Emission QYs were measured using a literature method with quinine sulfate (in 1.0 M sulfuric acid, Φr = 0.546) as a reference.30 Excited state lifetime was determined by an Edinburgh Instruments F920 spectrometer.

2.2 Synthesis of Triphenylamine-Derived Dyes Tn (n = 1–5)

4-(Diphenylamino)benzaldehyde was prepared following a literature method using triphenylamine as the starting material in the presence of DMF (N,N-dimethylformamide) and POCl3.31 Then, the synthesized 4-(diphenylamino)benzaldehyde (20 mmol) was dissolved in ethanol (40 mL), followed by the slow addition of a mixture of hydrazine hydrate and ethanol (20 mL:20 mL).32 The resulting solution was stirred at room temperature for 30 min and then at 50 °C for another 8 h. The resulting solid product was collected, washed with ethanol, and dissolved in ethanol (40 mL). To the final solution, the aldehyde compound (20 mmol) was added. The resulting deep red solution was stirred at 75 °C for 10 h. After extracting the solvent under reduced pressure, a brown-yellow solid was collected and purified on an Al2O3 column (FCP 100–200) with petroleum ether/acetic ether (v/v = 100:5) as the eluent. The detailed characterization of T1–T5 can be found in Supporting Information.

2.3 Cu2+ and CO Sensing Experiment of Tn (n = 1–5) via Absorption and Emission Spectra

For the sensing experiment, a mixed solvent of CH3CN/H2O (v/v = 1:1) was applied to ensure good solubility of dyes and various testing reagents (salts, interfering species et al.), despite the fact that this mixed solvent might lead to the degradation or hydrolysis of the dyes, which was mentioned below in Section 3.4.3. Tn dyes were dissolved in CH3CN/H2O (v/v = 1:1), then CuCl2 was added and stirred for 2 min before recording the absorption and emission spectra using the aforementioned Shimadzu UV-3101PC spectrophotometer and Hitachi F-7000 fluorescence spectrophotometer (fluorescence mode, slit = 2 × 2 nm). CORM-3 was added and used as a CO source in this work, so that CO concentration could be controlled.33 No CO bubbles were used in this work to avoid safety threats to crew members and to prevent heterogeneous contact between the Tn solution and the CO stream, which might lead to unexpected and uncontrollable CO concentration distribution in the Tn solution.

2.4 Cell Experiment and Fluorescence Imaging Performance

The cell experiment of selected Tn dyes was conducted with HeLa cells, which first underwent cultivation, measurement, and placement into a 96-well plate for cell culture. Afterward, they were subjected to a 24 h incubation at 37 °C with or without Tn; Tn&Cu2+; and Tn&Cu2+&CORM-3 (with a molar ratio of 1:1:10). Following this, the cells underwent staining using the CCK-8 assay at a wavelength of 450 nm to assess cytotoxicity. Optical density measurements were employed, and Cu2+ was introduced as CuCl2.

The performance of selected Tn dyes in the presence of an external CO source, CORM-3, was evaluated. Initially, HeLa cells underwent culture and incubation with Tn&Cu2+ (50 μM:50 μM in PBS) for 48 h. Following a PBS wash to eliminate residual Tn&Cu2+, cells were exposed to varying concentrations of CORM-3 (10, 50, and 100 μM) and cultured for 15 min. Subsequently, cells were washed with PBS and subjected to imaging using the Olympus FV1200-MPE spectral confocal multiphoton microscope mentioned earlier. A control experiment was conducted where only Tn&Cu2+ was used but no CORM-3 was added.

The endogenous CO imaging performance of selected Tn dyes was evaluated following the procedure described below, which utilized Heme as an internal CO producer. Initially, HeLa cells underwent culture with Heme (100 μM), followed by treatment with Tn&Cu2+ (1:1) for 15 min.34 Subsequent to incubation, the cells were rinsed with PBS, and images were captured using the previously mentioned Olympus FV1200-MPE spectral confocal multiphoton microscope.

3 Results and Discussion

3.1 Synthesis and Structure of Triphenylamine-Derived Dyes Tn (n = 1–5)

As aforementioned, to weaken the π–π attraction between the coplanar conjugation planes in pyrene-like molecules, triphenylamine was used as the structural core since its three phenylamine rings are noncoplanar with each other, which may partially decrease the possibility of forming excimer or exciplex structures. Then, aromatic rings having various substituents (—OH, —OMe, or —NMe2) are bonded to the triphenylamine core with an =N—N= group so that the correlation between geometric/electronic substituents and PL/sensing performance can be revealed.

Single crystals were obtained for T2, T3, T4, and T5, but not for T1. The unsuccessful preparation of a T1 single crystal may be explained by the missing phenolic hydroxyl group in T1, compared to that in T2, T3, T4, and T5, since a phenolic hydroxyl group increases the molecular dipole moment and thus helps to crystallize. It is observed from Figure 1 that one phenylamine ring from the triphenylamine is nearly coplanar with the aromatic ring, forming a conjugation plane, but the other two phenylamine rings of the triphenylamine are noncoplanar with this conjugation plane. Thus, the size of the conjugation plane in T1–T5 is controlled, weakening the π–π attraction between the coplanar conjugation planes and decreasing the possibility of forming excimer or exciplex structures. This statement is confirmed by the crystal stacking of T2, T3, T4, and T5. As shown in Figure S1 (Supporting Information), π–π stacking is observed in the T2 crystal but not in the T3, T4, and T5 crystals.

Figure 1 Single-crystal structures of T2 (a), T3 (b), T4 (c), and T5 (d). H atoms were omitted for clarity. See Supporting Information for full geometric parameters.

3.2 PET Revealed by Density Functional Theory Calculation in Tn (n = 2–5)

It has been reported by Hong and co-workers that there is PET (photoinduced energy transfer) in azobenzene derivatives, and their electronic transitions are sensitive to surrounding factors, such as geometric distortion and coordination with metal ions, showing changes in absorption wavelength, fluorescence wavelength, or intensity. This endows azobenzene derivatives with versatile possibilities of being a probe.25,26 The first 50 singlet electronic transitions of Tn (n = 2–5) were calculated by TD-DFT at the RB3LYP/6-31G(d) level using their single-crystal structures as the initial geometry. Based on these electronic transitions, their absorption spectra are simulated and compared to their experimentally measured spectra in Figure 2. Two selected orbitals, highest occupied molecular orbital (HOMO) and LUMO (lowest unoccupied molecular orbital), of each Tn (n = 2–5) molecule are plotted and shown in Figure 2. The HOMO of Tn generally consists of its triphenylamine group, while its LUMO is composed of the =N—N=C—Ar group. The onset of electronic transition from HOMO to LUMO thus has an obvious electron transfer character. Having an electron-donating group —N(Et)2 in its aromatic ring, T5 shows the highest transition electric dipole moment value (T2 = 20.676, see Supporting Information) among these four Tn (n = 2–5) molecules. While the —OMe group in T4 slightly decreases its transition electric dipole moment value (T2 = 11.546, see Supporting Information) compared to those of T2 (T2 = 13.371, see Supporting Information) and T3 (T2 = 12.823, see Supporting Information). It is thus concluded that the —N(Et)2 group increases the electron density of frontier molecular orbitals, which facilitates the PET transition between HOMO and LUMO. But the —OMe group decreases the electron density of frontier molecular orbitals and thus weakens the PET transition between HOMO and LUMO. Upon a structural distortion or transformation, such as coordinating with a metal center, the PET of Tn may be affected, showing spectroscopic signals for optical sensing. This statement will be later confirmed by the experimental result.

Figure 2 Frontier molecular orbitals of T2 (a, HOMO; b, LUMO), T3 (c, HOMO; d, LUMO), T4 (e, HOMO; f, LUMO), and T5 (g, HOMO; h, LUMO), and their simulated absorption spectra (i) calculated at RB3LYP/6-31G(d) level in vacuum (see Supporting Information for singlet electronic transition result).

3.3 Absorption, Emission, and QY of Intrinsic Tn (n = 1–5)

The photophysical parameters of Tn (n = 1–5) are determined and compared in Table 1, including absorption peaks (λabs), emission peaks (λem), and emission QYs (ϕ). Figure 3 shows the absorption spectra of Tn (n = 1–5) (in CH3CN/H2O, v/v = 1:1, 2 μM). Due to the rather similar molecular structures of Tn (n = 1–5), these absorption spectra are similar to each other in terms of wavelength and band shape. A slight difference is observed in terms of absorbance. Their multiple absorption bands are comparable to the simulated absorption spectra of Tn (n = 2–5), as shown in Figure 3. Thus, the absorption band of each Tn dye at ∼410 nm is attributed to PET absorption. The PET absorption of T1 is at 404 nm. The incorporation of electron donors (in T2, T4, and T5) or the increased size of the conjugation system (in T3) leads to a red shift of the PET absorption, as shown in Table 1. T5 has the highest absorbance among the five Tn dyes, and this result is consistent with its higher transition electric dipole moment value (T2 = 20.676, see Supporting Information) than those of other Tn (n = 2–5) molecules, as aforementioned in Section 3.2. While the other two absorption bands of each absorption spectrum at ∼340 and ∼230 nm are mostly preserved, this result means that the PET transition is sensitive to environmental variations and thus can be developed for sensing purpose.

Table 1 Photophysical Parameters of Tn (n = 1–5)

 	λabs (nm)a	respond tob	λem (nm)	fwhm (nm)	ϕ (%)	ϕ′c	ϕ″d	Trec (s)e	
T1	404	Cu2+, Fe2+, Fe3+, Hg2+, Ni2+, Zn2+, CO32–, PO43–, Cys, Hcy	539	91	3.5	1.8	2.2	386	
T2	413	Cu2+	550	72	5.4	0.7	1.6	378	
T3	421	Cu2+, Fe3+, Hg2+	558	87	4.7	1.8	2.4	403	
T4	415	Cu2+, Fe2+, Fe3+, Hg2+, Ni2+, Zn2+, CO32–, PO43–, Cys, Hcy	547	73	6.8	0.5	5.6	293	
T5	414	Cu2+	501	49	17.1	0.5	16.8	342	
a PET absorption.

b Justified by absorption variation, see Section 3.4.1.

c With the presence of Cu2+(1 equiv).

d With Cu2+(1 equiv) and CORM-3 (10 equiv).

e Determined by the time of reaching steady emission (intensity variation <2% within 30 s).

Figure 3 Absorption and normalized PL spectra of Tn in CH3CN/H2O (v/v = 1:1, 5 μM).

Figure 3 shows the emission spectra of Tn (n = 1–5) (in CH3CN/H2O, v/v = 1:1, 2 μM) and corresponding QYs, along with fwhm (full width at half-maximum) values, which are listed in Table 1. A Gaussian-type single-emission band is observed for each Tn dye. T1 shows a weak emission peaking at 539 nm with QY of 3.5%. The electron donors in T2–T4 shift their emission to longer wavelengths, with QYs slightly increased. The fwhm values of T1–T4 are >70 nm, indicating an intense structural relaxation in their excited state, decreasing their emissive energy content and quenching the emissive state, which is confirmed by their limited QYs (lower than 7%). As for T5, its electron donor [—N(Et)2 group] increases its QY to 17.1%, compared to T1 (QY = ϕ = 3.5%). However, the emission of T5 is blue-shifted to 501 nm with fwhm decreased to 49 nm, compared to T1 (λem = 539 nm, fwhm = 91 nm), which is consistent with the trend observed in T2–T4. There may be two reasons for the blue-shifted emission of T5 with decreased fwhm and increased QY: one is the decreased energy loss in the excited state, suggested by the decreased fwhm value of T5 (compared to T1), and the other is its high transition electric dipole moment, which allows for an efficient and fast emissive decay of the excited state, suppressing the nonemissive decay (energy loss) of the excited state.

3.4 Sensing Behavior of Tn (n = 1–5) Revealed by Absorption and Emission

3.4.1 Absorption Spectra in the Presence of Cu2+ and Competing Species: Selectivity

To obtain a fast evaluation of their sensing selectivity, the absorption spectra of Tn (n = 1–5) in the presence of metal ions, cations, and competing interferents are shown in Figure 4, including Fe2+, Fe3+, Hg2+, Na+, K+, Mg2+, Ca2+, Cd2+, Ni2+, Zn2+, Co2+, Mn2+, Sn2+, Cl–, Br–, S2–, SO42–, NO3–, CO32–, PO43–, Ac–, ClO–, glucose, GSH (l-glutathione), Cys (cysteine), and Hcy (homocysteine). Generally speaking, all five Tn dyes are sensitive to Cu2+, with their PET absorption blue-shifted from 410 to 420 nm to 380–390 nm. In addition, the secondary absorption band of free Tn dyes at 330–340 nm disappears after adding Cu2+. This absorption band is assigned as the electronic transition from n (Cl) to π* (aromatic ring).35,36 After adding Cu2+, the electron pairs of N atoms coordinated to Cu2+ (d orbitals), which explains the disappearance of this absorption band. T2 and T5 showed good selectivity since their absorption was blue-shifted by only Cu2+. As for T3, transition metal ions of Fe3+ and Hg2+ lead to an absorption blue shift as Cu2+ does. As for T1 and T4, their absorption blue shift can be triggered by more transition metal ions, anions, and amino acids. Generally speaking, T1 has the worst selectivity since it has no geometric or electronic factors to limit ion sensing. The electron donor in Tn improves sensing selectivity, which may be explained by the increased coordination affinity between the ion and Tn, resulting in good selectivities of T2 and T5. As for T3, its enlarged size of the conjugation plane, compared to T2, spreads the electron distribution and thus weakens the coordination affinity between the ion and T2, showing worse selectivity. As for T4, there may be an H-bond between the —OMe group and the —OH group, which weakens the recognition of —OH for metal ions.

Figure 4 Absorption spectra of T1 (a), T2 (b), T3 (c), T4 (d), and T5 (e) in CH3CN/H2O (v/v = 1:1, 10 μM) upon the presence of Cu2+ (10 μM) and interferents (10 μM) and corresponding PET absorbance variation (f). 0, Cu2+; 1, blank; 2, Fe2+; 3, Fe3+; 4, Hg2+; 5, Na+; 6, K+; 7, Mg2+; 8, Ca2+; 9, Cd2+; 10, Ni2+; 11, Zn2+; 12, Co2+; 13, Mn2+; 14, Sn2+; 15, Cl–; 16, Br–; 17, S2–; 18, SO42–; 19, NO3–; 20, CO32–; 21, PO43–; 22, Ac–; 23, ClO–; 24, glucose; 25, GSH; 26, Cys; and 27, Hcy.

3.4.2 Emission Quenching by Cu2+: Spectra and Mechanism

It has been found from Table 1 that Tn (n = 1–5) dyes all respond to the same analyte, Cu2+. Thus, we select Cu2+ as a quencher to discuss its emission quenching behavior. Figure 5 shows the emission spectra of Tn (n = 1–5, 10 μM) with increasing Cu2+ concentrations (0–1 equiv). It is observed that all five Tn dyes respond to Cu2+ by showing a decreased fluorescence intensity and QYs, as depicted in Figure 5 and Table 1.

Figure 5 Emission spectra of T1 (a), T2 (b), T3 (c), T4 (d), and T5 (e) in CH3CN/H2O (v/v = 1:1, 10 μM) upon increasing Cu2+ concentrations (0–10 μM); insets: emission intensity variation against [Cu2+] of Tn. Stern–Volmer plots of Tn (f) upon increasing Cu2+ concentrations (0–10 μM); inset: excited state lifetime of T5 upon Cu2+ concentrations of 0, 5, and 10 μM.

As for T1, T2, T4, and T5, their emission intensity simply decreases with increasing Cu2+ concentrations, showing no obvious spectral (blue or red) shift. No shoulder bands or new bands are observed. This result actually suggests a static quenching mechanism: Tn and Cu2+ form a nonfluorescent adduct, which decreases the amount of free/fluorescent Tn molecules, leading to a decreased emission intensity. More explanation words are needed for T3 since it shows a new emission band peaking at 644 nm in the presence of Cu2+ (>0.2 equiv). We attribute this emission to the adduct between T3 and Cu2+. Apparently, the naphthalene ring in T3 stabilizes the adduct between T3 and Cu2+ and, thus, shows an emission at 644 nm. The other four dyes, T1, T2, T4, and T5, fail to form a stable and emissive adduct with Cu2+. It is still observed that the linear quenching region of T4 (0–8 μM) is narrower than those of other four dyes (0–10 μM). A possible reason is that the —OMe group in T4 offers an additional bonding affinity with Cu2+, especially when Cu2+ concentration is high, and thus leads to the nonlinear quenching behavior.

To reveal the sensing mechanism of Tn, the excited state lifetimes (τ) of T5 in the presence of various Cu2+ concentrations are compared and are shown in Figure 5. A monoexponential decay mode is observed for T5 without Cu2+ with a lifetime of 7.1 ns. After increasing the Cu2+ concentration to 0.5 and 1.0 equiv, the lifetime is slightly decreased to 6.5 and 6.1 ns. Thus, combined with the aforementioned absorption variation caused by Cu2+, it is concluded that Tn molecules follow a static sensing mechanism toward Cu2+.

After confirming the static sensing mechanism, the fluorescence intensity variation of Tn against Cu2+ concentration is analyzed with the Stern–Volmer method.37 If a luminescent probe follows a dynamic sensing mechanism, its emission intensity (F) should follow a linear correlation against quencher concentration [quencher] with a slope of Ksv. Here, the subscript 0 means no quencher, Ksv is the quenching coefficient, and C is a constant.1

However, this is not the case shown in Figure 5. Nonlinear curves are observed. On the other hand, the fluorescence intensity of Tn is found to be inversely proportional to the Cu2+ concentration. This observation thus finally confirms a static sensing mechanism in Tn toward Cu2+, where Tn forms an adduct with Cu2+ and becomes nonfluorescent, leading to a decreased emission intensity.

3.4.3 Emission Stability upon Various Solvents and pH Values

To get a primitive understanding of the emission stability of these fluorescent dyes, the emission spectra of T1–T5 are recorded and compared to those after being aged for 7 days in various solvents (CH2Cl2, DMF, EtOH, and CH3CN/H2O). It is observed from Figure S2 and Table S1 (Supporting Information) that these dyes exhibit an emission red shift after being aged for 7 days, which shall be attributed to the degradation or hydrolysis of the Tn molecules. This red shift tendency is slight in CH2Cl2, but it is more obvious in CH3CN/H2O, which may be caused by the H2O molecules that accelerate the hydrolysis or degradation procedures of these dyes. To avoid such hydrolysis and/or degradation procedures, these dyes should be stored in solid state. A low temperature helps to restrain/decrease the hydrolysis and/or degradation procedures, as shown in Figure S3 (Supporting Information). As for the pH effect, T5 is selected as an example. It is observed from Figure S4 (Supporting Information) that an acidic condition (pH 5.8–7.0) quenches the emission intensity of T5 and leads to emission blue shift, which originates from the protonation of T5. While, with the pH value increased to 7.5, the emission intensity of T5 is increased, compared to that under pH 7.0, along with an emission red shift, which originates from the deprotonation of T5 (Ar—OH). An even higher pH value of 8.0, however, tends to quench the emission intensity of T5. The deprotonation procedure increases the electronic density of T5 and thus leads to an emission red shift. In the meanwhile, the deprotonated Ar—OH group of T5 may have severe geometric relaxation, leading to a decreased emission intensity at pH 8.0.

3.5 Emission Recovery of Tn&Cu2+ by CORM-3: CO Sensing Potential and Mechanism

It has been aforementioned that Tn forms an adduct with Cu2+ and becomes nonfluorescent. Bearing this sensing mechanism in mind, it is assumed that the quenched emission of Tn (in the presence of Cu2+) may be recovered if Cu2+ can be removed or replaced. In this work, a carbon monoxide releaser of CORM-3 was introduced, hoping to reduce Cu2+ to Cu+ and thus recover Tn emission.22Figure 6 shows emission spectra of Tn&Cu2+ (n = 1–5) after adding CORM-3, and their resulting QYs are determined and listed in Table 1. No surprise, Tn emission is quenched by Cu2+, then, after adding a CO releaser of CORM-3, Tn emission is recovered. The recovered QYs are determined and are listed in Table 1. As for T1, T2, and T3, their recovered emission spectra are similar to their intrinsic emission spectra, with no spectral shifts or new bands, but their recovered QYs are obviously lower than their intrinsic QYs (before adding Cu2+). As for T4 and T5, their recovered QYs are close to their intrinsic QYs (before adding Cu2+), and their recovered emission spectra are rather similar to those of their intrinsic emission spectra. This result suggests a reversible formation and/or dissociation of the adduct between T4/T5 and Cu2+. The difference in emission recovery between T1/T2/T3 and T4/T5 is tentatively attributed to the electron donors in T4 and T5.

Figure 6 Emission spectra of intrinsic Tn, Tn&Cu2+ (n = 1–5), and Tn&Cu2+ (n = 1–5) after adding CORM-3, T1 (a), T2 (b), T3 (c), T4 (d), and T5 (e). Emission intensity monitoring of Tn&Cu2+ (n = 1–5) after adding CORM-3 (f).

The time-resolved emission recovery of Tn (n = 1–5) is revealed by monitoring their emission intensity after adding CORM-3. As shown in Figure 6, a low emission intensity is observed for each Tn&Cu2+ sample. After the addition of CORM-3, the emission intensity increases instantly and finally remains stable. For convenience of comparison, the recovery time (Trec) is defined as the time for each Tn&Cu2+ sample to reach steady emission (intensity variation <2% within 30 s). Generally, it is found that the electron donors in T2, T3, and T5 decrease their emission recovery time compared to T1. T3 shows the longest recovery time among the five Tn dyes, which may be attributed to the steric hindrance of the anthracene group.

For a better understanding of the formation of Tn and Cu2+, two dyes (T2 and T5), which have different recovery behaviors, are selected, and the complexation constant (K) of T2&Cu2+ is calculated by eq 2 and compared with that of T5&Cu2+. The total concentration of T2/T5 and Cu2+ is fixed as 10 μM, and an absorption titration is performed.38 Here, A is the recorded maximum absorbance, A′ is the theoretical absorbance, and α is dissociation ratio.2

3

4

It is observed from Figure 7 that the absorbance of T2 and T5 reaches the maximum value at a [Cu2+]/{[Cu2+] + [Tn]} molar ratio of 50%, which means a 1:1 stoichiometric ratio between Cu2+ and T2/T5. The K value of T2 is determined as 1.635 × 109 L/mol, while that of T5 is 2.970 × 107 L/mol. Apparently, T2 has a higher coordination affinity with Cu2+ and forms a more stable adduct than T5. As a consequence, T2 shows poor emission recovery after adding CORM-3, but T5 shows good emission recovery after adding CORM-3.

Figure 7 Absorption titration spectra of T2 (a) and T5 (b). XPS spectra (c) and EPR result (d) of T5&Cu2+ (1:1) and T5&Cu2+&CORM-3 (1:1:10).

The emission recovery mechanism of Tn&Cu2+ by CORM-3 is tentatively discussed by a comparative XPS study. It is observed from Figure 7 that T5&Cu2+ renders an XPS peak of 934.0 eV, corresponding to Cu2+, along with a shoulder XPS peak of 933.0 eV corresponding to Cu2+, as previously reported by a literature.39 After the addition of CORM-3, the XPS intensity at 934.0 eV disappears, while that at 933.0 eV is clearly observed. This result suggests that Cu2+ has been reduced to Cu2+ by CORM-3. This statement is further confirmed by a comparative EPR (electron paramagnetic resonance) study. As shown in Figure 7, a typical EPR signal is observed for T5&Cu2+, which matches the empty d orbitals (d9) in Cu2+.40 After the addition of CORM-3, the EPR signal disappears, indicating the full d10 orbitals in Cu+.

3.6 Cytotoxicity and Exogenous and Endogenous CO Imaging with T5

After comparison of the five Tn dyes in terms of selectivity, fluorescence QYs, and emission recovery, T5 is found to be superior to the other four dyes and is thus selected for the following cell experiment. The disadvantages of T1, T3, and T4 are their poor selectivity, while T2 shows poor emission recovery in the presence of CO. T5 has good selectivity and obvious change of QYs (with Cu2+ or with CO) due to its suitable size of conjugation plane and substituents [—OH and —N(Et)2]. First, its cell viability testing is performed to evaluate its potential in bioimaging. As shown in Figure 8, a cell viability rate of 98.0% is observed in the presence of 10 μM T5 and then decreases with increasing T5 concentrations. At a T5 concentration of 100 μM, the cell viability decreased to 63.2%.

Figure 8 Cell cytotoxicity of T5 (a), bioimaging photos of HeLa cells without and with CORM-3 (b, 10; c, 30; and d, 100 μM), and HeLa cells with Heme upon various hours (e, 0.5; f, 1; and g, 2 h).

By addition of Cu2+ and CORM-3, the cell viability further drops to 56.1% (with Cu2+, 1:1) and 52.4% (with Cu2+ and CORM-3, 1:1:10), respectively. For the following experiment, T5 concentration is fixed as 50 μM to ensure cell viability. Then, the exogenous CO imaging performance of T5 is discussed using HeLa cells and CORM-3 as a CO source. No detectable emission is observed when only T5&Cu2+ is added. By increasing the CORM-3 concentrations to 20, 50, and 100 μM, the green emission becomes stronger and stronger, indicating a cell endocytosis process for CORM-3. Thus, exogenous CO imaging using CORM-3 has been confirmed. Then, using Heme as the endogenous CO source, the CO imaging performance of T5 is further revealed as follows. In the absence of Heme, no detectable emission signal is observed. After adding Heme (100 μM) to HeLa cells, the Heme metabolic process releases CO for T5 imaging.26 It is observed from Figure 8 that the green emission becomes stronger and stronger as the reaction time increases, confirming the endogenous CO imaging of T5 with the help of Cu2+.

4 Conclusions

In summary, this work reported five probes derived from triphenylamine with various substituents [—OH, —OMe, or —N(Et)2], including their synthesis, geometric structure, electronic transitions, comparison of photophysical properties (with or without Cu2+), and potential application in bioimaging. It was found that the electron donors in these probes improved selectivity and emission QY. T5 showed superior performance over the other four dyes. Its QY was quenched from 17.1 to 0.5% by Cu2+ and recovered to 16.8% after an exposure to CO within 342 s. T5 followed a static sensing mechanism by forming a nonfluorescent adduct with Cu2+. CO reduced Cu2+ to Cu+ and, thus, recovered T5 emission. The bioimaging performance of the optimal probe was evaluated as well. The novelty of these triphenylamine-based probes was that they showed no obvious influence from intermolecular π–π attraction, compared to the pyrene-based probes. The disadvantage of T5 was its unsatisfactory cell cytotoxicity. For future efforts, more similar probes with low cell cytotoxicity should be synthesized and tested. In addition, these fluorescent dyes in their present form are not suitable for gaseous CO sensing since they are dispersed in aqueous-based solutions or in condensed state. In aqueous-based solutions, the gaseous CO has a rather low solubility (0.0026 g/100 g), which makes the CO sensing in aqueous-based solutions meaningless (more or less). In condensed state, the gaseous CO can hardly penetrate the dyes in solid state, leading to sensing behavior only on solid surface and thus poor sensing performance. To apply these dyes for the sensing of gaseous CO, they should be dispersed or embedded or immobilized in a solid but porous host, which allows the fluent transportation of gaseous CO.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c03137.Characterization details and 1H NMR spectra of T1–T5; photo of T5 upon excitation wavelength of 414 nm; emission spectra of T1–T5 before and after being aged for 7 days in various solvents; emission spectra of T5 before and after being stored for 7 days at 4 and 35 °C; emission spectra of T5 in CH3CN/H2O solutions (10 μM) with various pH values (5.8–8.0); TD-DFT result of T2-T5 calculated at RB3LYP/6-31G(d) level in vacuum; packing mode of T2–T5 crystals; and crystal details and full geometric parameters of T2–T5 (PDF)

Supplementary Material

ao4c03137_si_001.pdf

The authors declare no competing financial interest.

Acknowledgments

The authors thank the financial support from the below funding, including the Natural Science Basic Research Program of Shaanxi (program no. 2021JQ-881), Medical Science and Technology Program of Henan (program no. SBGJ202303051), and Science and Technology Planning Project of Nanyang (program no. JCQY021).
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