A Poly(carbazole-alt-triazole) with Thiabendazole Side Groups as an “On-Off-On” Fluorescent Probe for Detection of Cu(II) Ion and Cysteine

A novel conjugated polymer PCZBTA-TBZ containing thiabendazole as recognition unit was synthesized via Suzuki coupling reaction, and its structural characterization, spectroscopic analysis and photophysical properties were investigated. In the metal ion response study, the addition of Cu2+ led to the occurrence of the photoinduced electron transfer (PET) mechanism, which significantly quenched the fluorescence of the polymer PCZBTA-TBZ with a quenching effect of 98%. Furthermore, I- can significantly quench the fluorescence of the polymer, but other anions have no such effect. According to the density functional theory calculation, compared with other polycarbazoles or other alternative copolymers containing carbazole, with alternating carbazole and triazole enhances the electron mobility and reduces the energy band gap of the polymer. Due to the strong coordination ability between Cu2+ and Cys, the adding Cys competes the Cu2+ in the [PCZBTA-TBZ-Cu2+] complex, blocking the occurrence of PET, and the fluorescence intensity of PCZBTA-TBZ is restored. The addition of other amino acids caused almost no change. The polymer is expected to be used for dual fluorescence detection of specific metal ions and Cys.


Introduction
Conjugated polymers (CPs) have been widely used in optoelectronics due to their excellent optical properties and remarkable semiconductor properties [1]. Since Swager and co-workers reported fluorescent chemical sensors based on conjugated polymers in 1995 [2], a large number of sensing systems have been developed using CPs as basic materials, which are used in detection for microbial pathogens [3], RNA [4], explosives [5], anions [6], metal ions [7], proteins [8], glucose [9] and enzymes [10]. In addition, due to their excellent detection efficiency, good physical properties, outstanding optoelectronic properties the high fluorescence quantum efficiency, and the chemical structure tunability based on combinatorial chemistry ideas, the CPs have become a research hotspot.
From the perspective of chemical structure, CPs with fluorescence sensing properties are mainly composed of two parts: main-chain and side-chain units that endow polymers with optical properties and certain functional groups that undertake the task of recognition and detection. The conjugated backbone determines the basic optical properties of 1 3 CPs and the recognition group on the side-chain or mainchain can enable the CPs to interact with the analyte, and the recognition ability such as machinability, sensitivity, detection limit, identification selectivity and immunity to interference of CPs can be further improved by different chemical modifications [11][12][13]. Compared with the small molecule fluorescent sensing materials, these conjugated polymers have better sensing properties and wider application fields due to the characteristics of the wires and the specific fluorescence amplification effect [14,15].
The fluorescence sensing systems of these conjugated polymers can be divided into several sensing mechanisms, such as PET, intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), excited state proton transfer (ESIPT) [16]. However, fluorescent sensing materials that rely on PET mechanism always have high sensitivity, selectivity and stronger stability, which makes PET mechanism the most common in chemical structure design [17]. The occurrence and efficiency of PET mechanism depends on the orbital energy level. The redox potential of the electron donor (D) and the electron acceptor (A) is the key factor to determine the orbital energy level [18].
At present, the fluorescent sensing materials constructed by metal ions and fluorescent small molecules have been applied in many fields [19,20]. These complex fluorescent sensing systems are usually based on an on-off or off-on detection strategy. A certain metal ion can quench or turn on the fluorescence of small molecules to form complexes. After adding the target detection substance, the fluorescence of the complex is restored or quenched. However, the composite sensing materials based on conjugated polymers and metal ions are rarely reported. Compared with small molecule composite sensing materials, more recognition and binding sites on conjugated polymers will endow better performance for the sensing systems, such as selectivity, high sensitivity and recyclability [21,22]. Based on this, the design of this kind of composite sensing system is very important. The current basic idea is to utilize the coordination or chemical reaction of metal ions with recognition units to facilitate the quenching and opening of fluorescent sensing systems.
It is well known that nitrogen-containing heterocycles have strong metal coordination ability. A large number of literatures have reported the introduction of aromatic N-heterocycles and their derivatives as recognition units into the main chain of conjugated polymers to coordinate with metal ions which leads to photophysical changes in the conjugated polymer [23,24]. Benzimidazole derivatives can be used as antibacterial, antiviral, insecticide and anticancer drugs [25], they often forming complexes with metal ions. Thiabendazole is a benzimidazole fungicide with weak autofluorescence. Due to the coexistence of benzimidazole and thiazolyl groups, these ligands can exhibit strong metal coordination ability and optimize the photophysical properties of CPs, so they are also widely used in N-containing heterocycles of photoelectric materials [26]. There are few reports about conjugated polymers containing thiabendazole, especially in the field of fluorescent sensing materials. Based on the above factors, we designed and synthesized a side-chain type conjugated polymer PCZBTA-TBZ via Suzuki coupling reaction. The main chain D-A alternating structure with a large π-conjugated rigid plane is composed of carbazole as electron donor and benzotriazole as electron acceptor, shortening the energy bandgap of the polymer and making the emission tend to the long wavelength region. At the same time, considering the importance of the recognition unit, thiabendazole was introduced as the recognition unit into the side chain through the alkylation reaction. Considering the relatively weak coordination ability of imidazole with copper ions, with the addition of cysteine, Cu 2+ can be deprived from the P-Cu 2+ complex, blocking the occurrence of PET mechanism and restoring the fluorescence [27]. We expect that PCZBTA-TBZ will provide an important reference for the development and utilization of novel conjugated polymer fluorescence sensors.

General Methods and Materials
1 H NMR and 13 C NMR measurements were carried out on a Bruker Avance II-400 MHz spectrometer in CDCl 3 with tetramethylsilane (TMS) as the internal standard. High resolution mass spectrometry (HRMS) spectra were recorded on a Q Exactive Plus. UV-Vis absorption spectra were obtained on an AnalytikJena Specord 200Plus spectrophotometer. The fluorescence spectra were recorded on an LS-55 luminescence spectrophotometer. All spectra were measured at room temperature. Molecular weight and molecular weight distribution were determined by gel permeation chromatography (GPC) by Waters GPC 2414 in THF using a calibration curve of polystyrene standards. The electrochemical characteristics of PCZBTA-TBZ (conc. 2 mg/mL) were studied by cyclic voltammetry (CV) in methylene dichloride on a CHI660 Electrochemical Workstation. Using a platinum button as working electrode, a platinum wire as counter electrode, an Ag/AgCl as reference electrode, and Tetra-n-butylammonium perchlorate (Bu 4 NClO 4 , 0.1 M) as supporting electrolyte.
All chemicals were purchased from Energy Chemical (Shanghai, China) and were used directly without further purification. Solvents were purified by standard procedures.

Calculation of Quantum Yield
The quantum yield was determined according to the following equation. Φ, F, A, n represents the quantum yield, the integrated area under the corrected emission spectra, the absorbance intensity at the excitation wavelength and the refractive index of solvent, respectively. In addition, refers to quinine sulfate in 0.1 N sulfuric acid (Φ = 55%, n = 1.33) as standard one [28]. PCZBTA-TBZ dissolved in THF solution (n = 1.40), s and u represent standard and target, respectively.

Polymer PCZBTA-TBZ
The polymer PCZBTA-TBZ was synthesized by Suzuki cross-coupling reaction. Under argon atmosphere, monomer 1 (0.1590 g, 0.3 mmol), monomer 2 (0.1680 g, 0.3 mmol), K 2 CO 3 (2 M, 2 mL) and two drops of methyltrioctyl ammonium chloride were added to toluene (8 mL) and stirred for 20 minutes after complete deoxygenation. Then Pd(OAc) 2 (0.0033 g, 0.0150 mmol) and tricyclohexylphosphine (0.0066 g, 0.0235 mmol) were added and the mixture was vigorously stirred at 90 °C for 24 h. After cooling to room temperature, the solution was poured into methanol (300 mL) and the precipitated polymer was filtered. The product was extracted with methanol for 48 h and dried under vacuum to afford a yellow solid (0.1193 g, 58.74% yield). 1

Fluorescence Sensing Experiment
In order to study the influence of anions and metal ions on the optical properties of PCZBTA-TBZ in solution, the PCZBTA-TBZ was prepared in a standard solution with a concentration of 5×10 -6 M (calculated by repeating unit), using THF as the solvent. Inorganic salts (I -, H 2 PO 4 -, Cl -, CH 3 COO -, HSO 3 -, NO 2 -, NO 3 -, SO 4 2-, F -, CO 3 2-, SCN -, HCO 3 and Br -) were dissolved in deionized water to afford a 3×10 -2 M solution and inorganic salts (Cu 2+ , Ag + , Fe 3+ , K + , Mg 2+ , Mn 2+ , Pb 2+ , Cd 2+ , Cr 3+ , Zn 2+ , Sn 2+ , Na + and Ca 2+ ) were dissolved in methanol to afford a 3×10 -3 M solution. Fluorescence titrations with excitation wavelength (413 nm) was carried out by adding aliquots of a solution of the selected inorganic salt to a THF solution of PCZBTA-TBZ in a quartz cuvette (1 cm width). For amino acids sensing analysis, selectivity was evaluated by adding an equal amount of Cysteine (Cys), Glutathione (GSH) and other various amino acids (Lys, Leu, Try, Ser, Thr, Glu, Phe, Ala, Gly, His, Tyr, Pro and Arg) to solution of [PCZBTA-TBZ-Cu 2+ ] probe independently and recording the fluorescence spectra of the corresponding mixed solutions. For the response time analysis, the solution of [PCZBTA-TBZ-Cu 2+ ] probe was added into various concentrations of Cys (8, 16, 24 and 32 µL, 1.0×10 -5 M) and the fluorescence spectra of the mixture were estimated after an incubation time of 0-10 min at 2-minute intervals.

Synthesis and Characterization
Compound 2 was characterized by 1 H NMR (Fig. S1), monomer 1 was characterized by 1 H NMR (Fig. S2), 13 C NMR (Fig. S3) and HRMS (Fig. S4), monomer 2 was characterized by 1 H NMR (Fig. S5) and 13 C NMR (Fig. S6), and the polymer PCZBTA-TBZ was analyzed by 1 H NMR (Fig. S7) and 13 C NMR (Fig. S8). A comparison of the 1 H NMR spectra of PCZBTA-TBZ and the monomers is shown in Fig. 1. The characteristic hydrogens of the polymer are in good agreement with their chemical structures. The low-field peaks (δ = 8.90-7.74 ppm) were attributed to the triazole ring of the carbazole ring and the aromatic hydrogen of thiabendazole, while the saturated hydrogens of the alkyl chain were all in the high-field (δ < 4.95 ppm). Among them, the 4.95-4.34 ppm signal is attributed to the hydrogen on the three methylene atoms directly connected to the nitrogen atoms of carbazole, triazole and thiabendazole, marked with H 9 . Monomer 1, monomer 2 in the spectra (δ = 4.80 ppm, δ = 4.64 ppm and δ = 4.36 ppm) correspond to three groups of triple peaks. At the same time, the unique single peak (δ = 1.40 ppm) of 24 hydrogen atoms on eight methyl groups of monomer 2 disappears in the 1 H NMR spectrum of PCZBTA-TBZ, indicating that the boronated group has been removed, confirming the Suzuki coupling the occurrence of the reaction. To sum up, PCZBTA-TBZ was successfully synthesized.
The molecular weight of PCZBTA-TBZ was determined by using GPC method. The M n value of the copolymer was 4846, the M w value was 10089, and the PDI was 2.08 ( Fig.  S9), indicating the conjugated polymer is more "purity", has better processing characteristics and more stable physical properties [32].

Basic Photophysical Properties of PCZBTA-TBZ
The absorption and emission spectra of PCZBTA-TBZ in different solvents at the same concentration (5×10 -6 M, calculated by repeating unit) are shown in Fig. 2, the spectroscopic properties of PCZBTA-TBZ did not vary strongly with solvent composition, and the absorption and emission maxima are listed in Table 1. PCZBTA-TBZ in various solutions showed two broad absorption bands in the range of 290-320 nm and 330-480 nm. The shorter wavelength absorption peaks come from the localized π-π * transitions, whereas the longer wavelength peaks may be attributed to the strong intermolecular charge transfer interaction between the carbazole and the triazole units [33]. The fluorescence spectrum of PCZBTA-TBZ have a peak at 471 nm in THF, exhibited quite strong blue-green light fluorescence with Stokes shifts of 57 nm, whereas in other solvents absorption band exhibited weaker than that in THF. However, the emission maxima of the polymers in DMSO and DMF are red-shifted to 483 nm and 477 nm, showing a photophysical-solvent dependency. In toluene, the polymer exhibits the largest Stokes shift of 85 nm, but considering the weak solubility of toluene for metal ions, and the polymer has a high fluorescence quantum efficiency (76.4%) in THF. Therefore, THF was selected as the solvent for subsequent fluorescence chemical sensing experiments.
In order to avoid a series of problems such as π-π * stacking between polymers at high concentrations and low fluorescence intensity at low concentrations, the emission spectra of PCZBTA-TBZ in THF solution of different concentrations are shown in the Fig. S10. When the concentration of PCZBTA-TBZ is 5×10 -4 M, its fluorescence emission intensity is the weakest. Then the π-π * stacking effect gradually weakened with the dilution of concentration, and the fluorescence intensity gradually increased accordingly.
When the solution was diluted to 5×10 -6 M, the polymer solution exhibited the strongest fluorescence emission intensity. In addition, with the dilution of the concentration, the maximum emission wavelength of the polymer solution is continuously blue-shifted. In summary, the PCZBTA-TBZ tetrahydrofuran solution with a concentration of 5×10 -6 M was selected as the experimental material for the next step of metal ion fluorescence.

Sensing Properties with Anions
To investigate the interaction between PCZBTA-TBZ and anions, the response characteristics of the polymer solution were studied by fluorescence spectroscopy in THF. As shown in Fig. 3a, when most anions were added, the fluorescence intensity of PCZBTA-TBZ had little change, while Iwas added, the fluorescence intensity decreases significantly. As shown in Fig. 3b, when the concentration of all anions is 5.8×10 -4 M (fluorescence quenching limit of I -), the fluorescence quenching degree of PCZBTA-TBZ with Iis 12.2, and others are about equal to 1.
As shown in Fig. 4, when other anions were added to the [PCZBTA-TBZ-I -] mixed solution, the fluorescence intensity of the mixed solution will hardly change, indicating that PCZBTA-TBZ has excellent anti-interference ability for the special recognition of Icompared with other anions.
In order to further study the response characteristics of the polymer to I -. As shown in Fig. 5a, the fluorescence intensity of PCZBTA-TBZ at 471 nm decreased significantly When the Iconcentration reaches 5.8×10 -4 M, and the quenching efficiency was 90%. Furthermore, we also calculated the fluorescence quenching efficiency according to the Stern-Volmer equation: where [A] is the concentration of analyte quencher, I and I 0 are the fluorescence intensity and the fluorescence intensity  at [A] = 0, respectively and K SV is the Stern-Volmer constant. As shown in Fig. 5b, when the concentration of Iis 5.8×10 -4 M, the interaction between polymer and Ireached equilibrium. And among low concentrations of I -(4.0×10 -4 M) a linear Stern-Volmer plot was obtained with the K SV value of 8.85×10 5 M -1 , correlation coefficients of R 2 = 0.9947. The above data show that PCZBTA-TBZ has special selectivity for Iand is not interfered by other anions, which may be caused by heavy-atom effect of Ion N-atom in carbazole ring [34].

Sensing Properties with Metal Ions
To examine the interaction between PCZBTA-TBZ and metal ions (6.6×10 -5 M), the responsive properties of PCZBTA-TBZ were studied by fluorescence spectroscopy in THF. As shown in Fig. 6a, the fluorescence intensity of PCZBTA-TBZ hardly changed when most of the metal ions were added, and decreased slightly when Ag + and Fe 3+ were added. In addition, Cu 2+ quenched the emission intensity of PCZBTA-TBZ by 98% at 471 nm, indicating that PCZBTA-TBZ has high specific selectivity to Cu 2+ compared with other metal ions. This high selectivity can be attributed to the high specificity of Cu 2+ and the stable enthalpy of complexation. Compared with other metals, the paramagnetism of Cu 2+ reduces the electron donor group. After coordination with Cu 2+ , it inhibits the internal charge transfer of PCZ-BTA-TBZ, thus inhibiting the occurrence of PET mechanism [35,36]. As shown in Fig. 6b, when the concentration of all metal ions is 6.6×10 -5 M which is the fluorescence quenching extreme limit of Cu 2+ , the fluorescence quenching degrees of PCZBTA-TBZ with Fe 3+ and Ag + are 9.8 and 8.7 respectively, and the others are approximately equal to 1. In particular, the quenching ratio of the polymer with Cu 2+ can be as high as 94.5. The results suggest that PCZBTA-TBZ is a potential fluorescent probe for Cu 2+ with high selectivity.
The response characteristics of the polymers to these three ions were deeply studied. As shown in Fig. 7a, after the addition of 6.6×10 -5 M Cu 2+ , PCZBTA-TBZ showed a significant fluorescence intensity at 471 nm had declined from 913 to 9, and the quenching efficiency was 98%. In addition, Fig. 8 significantly shows that color and fluorescence intensity changes of PCZBTA-TBZ with the addition of various metal ions (6.6×10 -5 M). Furthermore, we also calculate the fluorescence quenching efficiency according to the Stern-Volmer equation. Respectively and K SV is the Stern-Volmer constant, the quenching efficiency increases  with the tendency of the polymer to bind to the quencher in solution. This association can occur through the formation of complexes between the polymer and the quencher (static quenching) or as a result of collisions between the photoluminescent macromolecule and the quencher (dynamic quenching) [37]. As shown in Fig. 7b, among low concentrations of Cu 2+ (0-2.7×10 -5 M) a linear Stern-Volmer plot was obtained with the K SV value of 5.47×10 5 M -1 , correlation coefficients of R 2 = 0.9947, and the curve generally shows an upward curve instead of a straight line with an intercept  Therefore, the fluorescence quenching of the polymer caused by Cu 2+ can be attributed to the static quenching of the formation of a complex with the strong coordination between the TBZ unit on the side chain and the Cu 2+ instead of a dynamic quenching process due to collision in activation [38]. The results imply that PCZBTA-TBZ is a promising fluorescence "turn off" chemical sensor for Cu 2+ with high sensitivity.
In addition, the fluorescence spectra of PCZBTA-TBZ (5.0×10 -6 M) after adding Ag + is shown in Fig. 9a. It can be seen that the fluorescence intensity of the polymer is significantly quenched from 913 to 99 with increasing Ag + concentration and the quenching efficiency was 89%. The Stern-Volmer plot of PCZBTA-TBZ is shown in Fig. 9b. When the Ag + concentration is 7.8×10 -5 M, the interaction between the polymer and Ag + reaches equilibrium. Ag + concentrations ranged from 0 to 5.5×10 -5 M, and the Stern-Volmer linear fit gave a K SV of 1.09×10 5 M -1 (R 2 = 0.9935).
Meanwhile, we can also see from Fig. 6a that PCZ-BTA-TBZ is selective for Fe 3+ . Therefore, we also performed the Fe 3+ titration experiment as shown in Fig. 10a. When 8.7×10 -5 M Fe 3+ was added, the fluorescence intensity of the polymer decreased from 913 to 87, and the quenching efficiency was 90%. Fig. 10b is the Stern-Volmer curve of PCZBTA-TBZ and Fe 3+ , which can be roughly regarded as a straight upward slope. When the Fe 3+ concentration is in the range of 0-5.0×10 -5 M, the fluorescence intensity changes linearly with the Fe 3+ concentration. At this time, the fitting degree R 2 = 0.9908, and the K SV is 7.65×10 4 M -1 . In addition, the detection limits of the polymers PCZ-BTA-TBZ for Cu 2+ , Ag + and Fe 3+ were calculated using the following formula: where LOD is the limit of detection, σ is the standard deviation of the experiment, and K SV is the quenching constant. The standard deviation of the known test is 0.007, the calculated detection limit of the polymer for Cu 2+ , Ag + and Fe 3+ are shown in Table 2. Obviously, PCZBTA-TBZ is more sensitive to the choice of Cu 2+ compared with Ag + and Fe 3+ . Table 3 lists previous reports of fluorescent detection of Cu 2+ ions using CPs for comparison. The newly developed polymer PCZBTA-TBZ showed better performance in terms of sensitivity, detection limit and selectivity. In addition, the sensing material has the advantages of simple preparation, low cost, and label-free detection.

Theoretical Calculation
In order to further explore the changes of properties of the target compound such as frontier orbital and energy level distribution. Monomer 1, monomer 2 and polymer molecules with chain length n=1 was theoretically calculated at the B3LYP/6-31G* density functional theory level using the Gaussian 09 software package. To save calculation time, methyl groups were chosen instead of octyl chains [42]. After optimizing the energy and structure of the above compounds, it is found that the minimum imaginary frequency is a positive number, indicating that the optimization is successful. As shown in Fig. 11, it can be found that the whole polymer molecule is in a large plane structure. This rigid large plane structure can effectively reduce the phenomenon of electron accumulation caused by structural distortion, and reduce the energy loss of electrons when flowing. It is not difficult to find from the calculation results that carbazole molecule has a high HOMO level and can be used as an electron donor, while benzotriazole has a low LUMO level and can be used as an electron acceptor. Therefore, when the polymer is in the ground state, the electron cloud is mainly concentrated on the carbazole, and once the polymer is excited, the electron cloud flows more to the benzotriazole. This D-A structure enhances the electron mobility, reduces the energy band gap of the polymer, and plays a certain energy band regulation effect [43].

Electrochemical Properties
The cyclic voltametric curve of PCZBTA-TBZ is shown in Fig. S11. From the onset oxidation potential (Eonset ox) and the onset reduction potential (Eonset red) of the polymer, the HOMO and LUMO energy levels as well as the electrochemical energy band gap (Eg, el) can be calculated according to the following equations:    PCZBTA-TBZ has superior air stability [44]. The electrochemical band gap is 1.36 eV, which is slightly smaller than the theoretical calculated value due to the normal error of the measurement method, may be caused by the decrease of the LOMO energy level due to the increase of the electron absorption capacity of the polymer with the increase of the chain length.

Response of the Complex of PCZBTA-TBZ and Metal Ions to Amino Acids
According to previous reports, Cys can combine with specific metal ions such as Ag + considering the strong coordination ability of thiols with specific metal ions such as Cu 2+ and Ag + [45,46], because thiol have a strong interaction with metal ions and form the M z+ -S (M z+ is Cu 2+ and Ag + ) bond. Therefore, bio thiols could eliminate the coupling effect between M z+ and PCZBTA-TBZ and recover the fluorescence of PCZBTA-TBZ in this system. The assumption is supported by Fig. 12. For detecting of bio thiols, Cys was added to the [PCZBTA-TBZ-M z+ ] complex solution, and the effect of other amino acids (Lys, Leu, Try, Ser, Thr, Glu, Phe, Ala, Gly, GSH, Tyr, Pro, Arg and His) were evaluated. As shown in Fig. 12 Cys led 67% fluorescence recovery at 417 nm, however, other amino acids only cause weak changes in fluorescence. This indicates that the complex [PCZBTA-TBZ-Cu 2+ ] has the highest sensitivity to Cys and can be used as a selective Cys chemical sensor. As shown in Fig. 13a, the fluorescence intensity of the complex solution was extremely weak. But after Cys is added, the fluorescence intensity increases gradually until 3.2×10 -4 M, the fluorescence intensity no longer changes. At this time, the fluorescence intensity of the polymer was enhanced from 8.9 to 603 with a 67-fold increase. Which demonstrates that Cys snatches the Cu 2+ in [PCZBTA-TBZ-Cu 2+ ] and form a new complex (Cys-Cu-Cys), releasing the polymer and thus restoring the fluorescence of the system. Because of the strong nucleophilicity of the S donor on the Cys molecule, Cys has a strong coordination ability with Cu 2+ ions, and a stable Cys-Cu-Cys complex is formed through the Cu-S bond. The linear relationship between the concentration of Cys and the fluorescence recovery intensity is shown in Fig.  S12. The detection of Cys was realized by the fluorescence signal "on-off-on".  In order to further study the detection of Cys by [PCZ-BTA-TBZ-Cu 2+ ], we investigated the fluorescence recovery kinetics using fluorescence spectroscopy. As shown in Fig. 13b, when Cys was added to [PCZBTA-TBZ-Cu 2+ ] solution, the fluorescence intensity increased immediately. When the reaction time reached 4 minutes, the fluorescence intensity hardly changed. These results show that Cys can complete the recovery process of fluorescence [PCZBTA-TBZ-Cu 2+ ] in a short time. With the extension of reaction time, the fluorescence intensity changes little and the recovery process is stable. Table 4 shows that this complex has a fast response speed.
In addition, competition experiments were also conducted to evaluate the practical applicability of the conjugated polymer complex sensor to Cys. Figure 14 shows the fluorescence intensity of other amino acids (3.2×10 -4 M) were added to the PCZBTA-TBZ solution, and the fluorescence intensity of Cys (3.2×10 -4 M) was added to the solution containing PCZBTA-TBZ and other amino acids. The results show thatα-amino acids hardly cause significant interference. In summary, the complex [PCZBTA-TBZ-Cu 2+ ] is a potential Cys fluorescence sensor.
The fluorescence intensity of [PCZBTA-TBZ-Ag + ] in the presence of Cys was measure, and the results are shown in Fig. S13.Not only does the presence of Cys restore the fluorescence intensity of [PCZBTA-TBZ-Ag + ], but also the presence of other amino acids restores the fluorescence intensity of [PCZBTA-TBZ-Ag + ] to a large extent, which may be attributed to Ag + and α-amino acids [50]. Figure 15a intuitively compares the influence of Cys on the fluorescence intensity about [PCZBTA-TBZ-M z+ ] complex. It is obvious that Cys cannot effectively restore the fluorescence of [PCZBTA-TBZ-Fe 3+ ] complex. At the same time, it can be seen from the competition experiment in Fig. 15b that the presence of other amino acids greatly interferes with the interaction of [PCZBTA-TBZ-Ag + -Cys]. All results illustrate that [PCZBTA-TBZ-Cu 2+ ]is an excellent sensing material for detecting Cys.

Conclusion
In conclusion, we designed and synthesized a novel conjugated polymer PCZBTA-TBZ with thiabendazole as the recognition unit through the Suzuki coupling reaction, which can detect Ag + , Fe 3+ and Cu 2+ based on the PET sensing mechanism. When the concentrations of Cu 2+ , Ag + and Fe 3+ were 6.6×10 -5 M, 7.8×10 -5 M and 8.7×10 -5 M, the fluorescence intensity of the polymer solution was quenched by 98.0%, 89.0% and 90.0%, respectively. The Stern-Volmer plots of PCZBTA-TBZ versus the three metal ions were linear at low concentrations with a K SV of 5.47×10 5 M -1 , 1.09×10 5 M -1 and 7.65×10 4 M -1 , respectively. When the concentration of Iis 5.8×10 -4 M, the fluorescence of polymer solution can be quenched by 90%. The Stern-Volmer plot of PCZ-BTA-TBZ versus the Iwas linear at low concentrations with a K SV of 8.85×10 5 M -1 . In the study of density functional theory, it was found that the D-A structure of the polymer main chain alternately composed of carbazole and triazole can well control the energy band of the polymer. Moreover, due to the stronger coordination ability of Cu 2+ with Cys, [PCZBTA-TBZ-Cu 2+ ] can achieve effective detection of Cys, because PCZBTA-TBZ is replaced by Cys to form a Cys-Cu-Cys complex and PCZ-BTA-TBZ, and the fluorescence is enhanced 67-foldwithout interference from other amino acids. Therefore, PCZBTA-TBZ is expected to become a sensing material for metal ions and Cys through fluorescence "on-off-on". Provides good ideas for the detection of cations and small molecules by indirect methods using conjugated polymer chemosensory. Finally, it provides an effective platform for the formation of complexes between conjugated polymers and metal ions using ligand replacement strategies to detect small organic molecules.  Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work is financially supported by the National University Student Innovation Program (no. 202110626042) and the 'Shuangzhi' Project of Sichuan Agricultural University (no. 00770105).

Data Availability
The datasets generated during and/or analyzed during the current study are available in manuscript file.

Declarations
Ethical Approval Not applicable.

Competing Interests
The authors declare no competing interests.