Synthesis of carbazole-based dendritic conjugated polymer: a dual channel optical probe for the detection of I− and Hg2+

ABSTRACT A new type of carbazole-based blue-emitting dendritic conjugated polymer, poly[(9,9-dioctyl)-2,7-fluorene-co-4,4’,4”-triphenylamine-co-9-(4-(9H-carbazol-9-yl)butyl)-3,6-carbazole](P), was successfully synthesized by Suzuki coupling reaction. Chemical structures of monomers and polymer were verified by FI-IR and 1HNMR characterizations. We found that polymer showed a special selectivity and high sensitivity for I−. With the addition of I−, the fluorescent polymer solution was obviously quenched. The polymer showed a special detection effect on I−. However, the fluorescent polymer was obviously restored when Hg2+ was added to the P/I− system due to the large complexation between I− and Hg2+. The anti-interference experiments of probe P/I− showed that other background cations have a slight influence on detecting Hg2+, and the calculated detection limit of Hg2+ reached 9.7 × 10−8 M, which could be a potential application for a two-channel cyclic detection of I− and Hg2+. Additionally, it was found that the theoretical values were in agreement with the experimental data.


Introduction
In recent years, heavy metal pollution has drawn much attention due to its irreversible negative effect on human health and the environment. However, the still widely used heavy metals in the cosmetics, industrial, and material fields accumulate in living organism and cause vital diseases to humans and animals as well as pose a serious threat to the natural environment [1,2]. Among the various heavy metal ions, mercury ions and their derivatives are listed as the deadly toxins and pollutants among the 'precarious dangerous pollutants' [3]. In the human body, mercury is accumulated through the skin, respiratory tract, food, and so on, and can cause digestive system diseases, muscle weakness, nerve disorders, and kidney disease [4,5]. Therefore, there is an urgent need to develop a mercury ion detection that can be easily and quickly applied.
In the past few decades, researchers have developed several methods to detect Hg 2+ , such as atomic absorption spectrometry [6], inductively coupled plasma mass spectrometry [7], anodic stripping voltammetry [8], and so on. Although these traditional detection methods have good sensitivity and selectivity, they are expensive, timeconsuming, complicated to operate and require professional operators, and not suitable for on-site detection. Therefore, after tremendous hard work from researchers, optical probe detection methods that can quickly and accurately detect metal ions in environmental and biological samples are coming out [9][10][11][12][13][14][15]. Recently, a novel La 3+ -BSA-AuNCs-based fluorescent probe was reported by Kailasa'group [16]. This probe can detect four divalent metal ions (Hg 2+ , Cu 2+ , Pb 2+ , and Cd 2+ ) simultaneously and exhibit excellent performance. Wang's group reported a quinoline-based derivative and its application for a sensitive probe detection of Hg 2+ and Iin biological fields [17]. Teng et al. reported green-emission carbon dots (CDs) as fluorescent off-on probes for the highly selective sensing Hg 2+ and I − [18].
Fluorescent conjugated polymers (CPs) are one of the optical probe organic materials [19][20][21][22][23], which show excellent sensitivity in ion recognition due to their optical signal amplification properties [24,25]. In 2020, Maimaiti yiminget al. designed and synthesized a pyrimidine group containing conjugated polymer, in which it showed excellent optical properties for specific recognition of Hg 2+ and I − [26]. Neha's group designed and synthesized a watersoluble polymer probe with a side chain containing rhodamine group, which was used to detect Hg 2+ in water and living cells and carry out naked eye colorimetry (colorless to pink to purple) for Hg 2+ [27].
The carbazole and its derivatives are an important class of nitrogen-containing heterocyclic compounds. It has a rigid molecular structure, good planarity, and biocompatibility as well as excellent optical properties [28][29][30]. Moreover, carbazole has a remarkable feature, which is easy to functionalize and covalently connect with other molecules, so it is widely used as a building block in various fields [31][32][33][34][35][36][37][38][39]. Recently, it has been reported that carbazole and triphenylamine-based conjugated polymer probes have certain advantages in the detection of I − and Hg 2+ [40][41][42]. Wang's group reported carbazole and triazolecontaining conjugated polymer as a visual and fluorometric probe for Hg 2+ and I − [43]. They showed excellent detection and recognition performance for Hg 2+ and I − . However, the dendritic fluorescent conjugated polymer probe containing the carbazole group has not been reported yet.

Instruments
IR spectrum was recorded on an EQUINOX 55 FT-IR spectrometer with KBr pellets. 1 HNMR and 13 C NMR spectra were collected on a BRUKER AVANCE III spectrometer. Number-average (M n ) and weightedaverage (M w ) molecular weights were determined by Waters GPC LC20AD. UV-visible absorption spectra were recorded on a SHIMADZU UV-2450 UV-vis spectrophotometer. PL spectra were recorded on RF-5301PC spectrophotometer and the test slit width was 5 nm × 5 nm. HRMS was recorded on Agilent qtof-6550.
All-electron DFT calculations have been carried out by the latest version of ORCA quantum chemistry software (Version 5.0.1) [45]. For geometry optimization calculations of S 1 structure, PBE0 functional [46] and the mixed basis set (SARC-DKH-TZVP basis set for I atom and the def2-SV (P) basis set for other atoms) were used, and the optimal S 1 geometry for each compound was determined [47,48]. The DFT-D3 dispersion correction with BJ-damping was applied to correct the weak interaction to improve the calculation accuracy [49,50]. The Conductor-like Continuum Polarization Model was used to account for the solvation effect of THF [51]. The excited states and Spin-Orbit Coupling (SOC) calculations were performed with B3LYP functional and the mixed basis set (SARC-DKH-TZVP basis set for I atom and DKH-def2-TZVP(-f) basis set for other atoms) [52].
The chemical structures of P were characterized by FT-IR and 1 H NMR characterization. As shown in Figure  S17, it can be found that the absorption peak at ~1141 cm −1 , corresponds to the stretching movement of the (C-N) group in tertiary amine. 1 H NMR spectrum of P was shown in Figure S18. It can be seen that the aromatic hydrogen proton peaks of the fluorene, carbazole, and triphenylamine groups appeared at ~ δ 8.523 ~ 7.069 ppm. Chemical shifts within the range of δ 4.374 ~ 4.165 ppm corresponded to the signal of the methylene (-CH 2 -), which was connected to the nitrogen of the carbazole. The chemical shifts range from δ 2.212 to 1.957 ppm corresponded to the hydrogen -CH 2 -in the fluorine and the hydrogen in the β-methylene group connected to the nitrogen of the carbazole. It can be seen that the 1 H NMR spectrum of P matches well with its chemical structure.

Thermal properties
In order to study the thermal stability of P, the thermogravimetric measurement of polymer probe P was conducted in the temperature range from 300 K to 650 K. As shown in Figure 1, by analyzing the thermogravimetric test curve of P, it could be found that polymer P began to decompose near 555 K and the structure began to fracture. This indicated that polymer P is stable enough before 555 K, which proves that the probe has good thermal stability.

Optical response of P toward I −
To explore the selectivity of polymer P to I − , spectral response tests of P (with the concentration of ~1.0 × 10 −5 M in THF) toward common anions were investigated. Figure 2a is the UV-vis absorption spectra of P in various common anions. As can be seen from Figure 2a, the absorption peak at 350 nm was significantly enhanced and the color of the solution changed from colorless to light yellow after I − was Scheme 1. Synthesis of monomers and polymer.   Figure 2b is the fluorescent emission spectra of P in various common anions. Also, it can be seen that the fluorescent intensity was significantly quenched when I − was added and other anionic ions just brought slight influence change.
The concentration titration experiments were also carried out. Figure S3 showed the UV-vis absorption spectra of P in the THF solution with the addition of I − . As shown in Figure S3, the UV-vis absorption spectra of P showed a monotonous increasing trend when the I − concentration was changed from 3.3 × 10 −7 M to 3.7 × 10 −4 M. When the I − concentration reached 3.7 × 10 −4 M, the UV-vis absorption spectra almost reached maxima. Figure S4 displayed the detailed fluorescent response of P toward I − . With the addition of I − , the fluorescent polymer P solution was quenched. The mechanism of fluorescence quenching in this experiment might be via 'heavy-atom' effect between the excited state of carbazole group and I − [40][41][42][43]. As can be seen from the illustration in Figure S4, it has a good linear relationship between polymer probe P and iodine ion concentration. The calculated detection limit of P to I − reached 6.2 × 10 −8 M by 3σ rule [53].
Response times of I − toward P were carried out, and the corresponding results are shown in Figure 3. It can be seen from Figure 3 that the fluorescent intensity of the P solution reached maxima after adding Iin 60 s. It showed that the detection of I − took place immediately. Competition experiments were also investigated, and the results are shown in Figure 4. It can be seen that other coexisting anions have little effect on the detection of probe P. When I − coexisted with these anions, the fluorescence was obviously quenched, which proved that P has excellent anti-interference for I − detection.

Optical response of P/I − composite probe to Hg 2+
Since I − and Hg 2+ have a strong binding effect, it is interesting to explore whether Hg 2+ was added to the polymer P/I − system to achieve changes in absorption and fluorescence intensity. Figure 5 shows the UV-vis spectra of various metal ions added to the P/I − system. As can be seen that after adding Hg 2+ to the P/I − system, the UV-vis absorption was significantly reduced. Meanwhile, the UV-vis absorption was only slightly reduced with the addition of Ag + . When other cations were added, the color of the polymer P/I − solution remained unchanged. It can be seen from Figure 6 that except Hg 2+ can restore the fluorescence of the composite system, other ions have no effect on the probe, which indicated the composite probe of P/I − was feasible for Hg 2+ detection with the naked eye. Figure 7a shows the fluorescent emission spectra of probe P after Hg 2+ stepwise titration. As shown in Figure 7a, when the concentration of Hg 2+ is (2.95 × 10 −4 M), the recovery of the fluorescent intensity has been slowed, and the fluorescent intensity has returned to 78% of the initial value. When Hg 2+ was dropped again, the fluorescent intensity was almost unchanged. The reason for this could be that the fluorescent quenching groups of HgI 2 and [HgI 4 ] 2existed in the polymer system [41][42][43]. After applying the 3σ rule [53], the calculated detection limit of the polymer P/I − system for Hg 2+ was 9.7 × 10 −8 M (Figure 7b).
In order to explore the anti-interference properties of the polymer P/I − composite probe, the anti-interference experiment of the polymer P/I − composite probe against Hg 2+ under background ions was carried out. As shown in Figure 8, P/I − only showed a strong recognition response to Hg 2+ , after addition of Hg 2+ to the polymer P/Iin the presence of other background ions, the fluorescent intensity of the polymer P/I − could almost be restored, so the polymer P/I − composite probe has a good detection for Hg 2+ with anti-interference.
The time response experiment of P/I − toward Hg 2+ was also studied. As shown in Figure 9, the time response properties were detected by fluorescence spectroscopy. The intensity of the composite probe P/  I − was basically stable within 1 min. As Hg 2+ was added to the probe system, the fluorescent intensity increased gradually after a period of time, and reached stability after 3 min. This showed that P/I − has good timeliness and is conducive to real-time detection.
The reversible cycle detection of P was investigated. It was found that the polymer probe P showed good reversible cycling in the first 12 cycles due to the alternating introduction of I − and Hg 2+ (as is shown in Figure 10). However, with the continuous addition of I − and Hg 2+ , the recovery of fluorescent intensity of polymer probe P was decreasing due to the increasing accumulation of HgI 2 or [HgI 4 ] 2-, which are quenching groups in the system.
Active materials-based test strip experiments were also carried out and represented practical utilization. The filter paper was soaked in the solution of P (1.0 × 10 −5 M) for a few seconds and dried naturally and the corresponding detection experiments were carried out. The pre-treated filter paper was then exposed to the solution of I − (0.1 M), and the fluorescent quenching of the test strip could be observed by naked eyes (as  is shown in Figure 11). When the Iexposed test strip was immersed into the Hg 2+ (0.01 M) solution, the fluorescent intensity increased significantly. This indicated that the conjugated polymer probe could be expected to be used in practice. The analytical performance of dendritic carbazole-based CP fluorescence method was compared with the reported methods for the detection of I − and Hg 2+ (Tables 2, 3). In contrast, although the linear range fitted for Iin this experimental method was relatively narrow, the linear range fitted for the detection of Hg 2+ by this method showed a wider linear range than that reported method in the literature [17,42,43,[59][60][61]. The calculated detection limit obtained by this experimental method was inferior to the reported detection method [17,27,55,56,58,60], and better than the reported method [18,42,43,53,54,57,59,61]. This method was comparable with the other analytical techniques [6,7], however, the other analytical techniques required high-cost instruments, professional operation, extensive sample pretreatment, inevitably rendering them unsuitable for real-time and in the field detection of I − and Hg 2+ .

Theoretical studies
In order to explore the detection mechanism of probe P, ORCA quantum chemistry software was used for electron DFT calculations. The corresponding orbit and energy level diagram and electron hole diagram are shown in Figure 12. Through B3LYP calculation, the energy level gap between HOMO and LUMO of structural unit of P before the addition of I − was 4.216 eV, and the energy and oscillator strength of S 0 to S 1 transition in S 1 stable structure were 3.585 eV and 0.06, respectively. Considering the influence of the spin-orbit coupling on fluorescence, it was found that there was no obvious coupling between S 1 and T X , and the oscillator strength was 0.0618. After adding iodine ion, the energy gap was 3.565 eV, and the energy and oscillator strength of S 0 to S 1 transition in S 1 stable structure were 2.898 eV and 0.01, respectively. In terms of oscillator strength, after adding of I − , the     fluorescent emission intensity of the system decreased to one-sixth of the original fluorescent intensity, and the oscillator strength has been reduced to the quenching level. This was consistent with our experimental values.

Conclusions
A new type of carbazole-based blue-emitting dendritic conjugated polymer P was successfully synthesized by the Suzuki coupling reaction and P showed good thermal stability before 555 K. Optical experiments showed that the polymer has specific selectivity, high sensitivity, and good anti-interference to I − . Meanwhile, P/I − complex can achieve the turn-on detection of high toxic Hg 2+ . The calculated detection limit of I − and Hg 2+ can reach 6.2 × 10 −8 M and 9.7 × 10 −8 M, respectively. Theoretical calculations were in agreement with the experimental data. Test strip experiments indicated that the conjugated polymer probe was expected to be used in practice. P was expected to be a potential optical probe material for two channel cyclic detections of I − and Hg 2+ .