A dual-analytes responsive ﬂ uorescent probe for discriminative detection of ClO − and N 2 H 4 in living cells

Hydrazine (N 2 H 4 ) and ClO − are very harmful for public health, hence it is important and necessary to monitor theminlivingcells.Herein,werationallydesignedandsynthesizedadual-analytesresponsive ﬂ uorescentsensor PTMQ for distinguishing detection of N 2 H 4 and ClO − . PTMQ underwent N 2 H 4 -induced double bond cleavage, affording colorimetric and green ﬂ uorescence enhancement with good selectivity and a low detection limit (89 nM). On the other hand, PTMQ underwent ClO − -induced sulfur oxidation and displayed red ﬂ uorescence lighting-up response towards ClO − with good selectivity, rapid response (<0.2 min) and a low detection limit (58 nM). Moreover, PTMQ was successfully employed for in-situ imaging of N 2 H 4 and ClO − in living cells. © 2020


Introduction
Hydrazine (N 2 H 4 ) is an essential chemical substance, which has been widely used in chemical synthesis and catalysis, as well as in the pharmaceutical industry [1,2].Hydrazine is generally produced by hypochlorite (ClO − )-mediated oxidation of ammonia or urea.Hence, the discharged industrial wastewater from hydrazine manufacture usually contains hydrazine and hypochlorite, which would be taken up by aquatic organisms/microorganisms and cause extremely toxic effect on them.Besides, N 2 H 4 can cause severe damage to the liver, kidneys, lungs, central nervous system, and the respiratory system.For this reason, N 2 H 4 has been categorized into a highly toxic and carcinogenic substance (B 2 grade) by US government [3], and the concentration of N 2 H 4 in water should be lower than 10 ppb [4].Therefore, it is of great importance to monitor hydrazine in living cells/organisms.
It is disclosed that integration of two different reaction/binding sites into a single fluorescent probe could be an efficient approach to achieve discriminative detection of two analytes [17,[26][27][28].In this study, we designed a dual-analytes responsive fluorescent probe PTMQ for the distinguishing detection of ClO − and N 2 H 4 .Probe PTMQ possesses two reaction sites: (1) the sulfur atom (\ \S\ \) located at the 9position of phenothiazine serves as a reaction site for ClO − ; (2) the MQ moiety acts as the reactive site for N 2 H 4 .We anticipated that PTMQ would separately react with HClO and N 2 H 4 , generating fluorescent products with different color/fluorescence signal output.We investigated the colorimetric and fluorescence responses, selectivity and sensitivity, detection limit, as well as time-dependent fluorescence

Chemicals and instrumentations
All chemicals were commercially purchased from Sigma-Aldrich and used directly without further purification.Compound 1 (N-ethyl-4methylquinolinium iodide, MQ) was prepared according to the literature [29]. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were measured on a Bruker AV spectrometer.High resolution mass spectrometry (HRMS) was obtained on HP-1100 LC-MS spectrometer.UV-Vis absorption spectra were performed on a Hitachi UV-3310 spectrometer.Fluorescence spectra were obtained with a FL-4500 fluorescence spectrometer.The cells imaging experiments were taken under a Nikon A1 confocal laser-scanning microscope with a 100 × objective lens.

Fluorescence imaging of ClO − and N 2 H 4 by PTMQ in HeLa cells
HeLa cells were seeded in glass-bottomed dishes and cultured in DMEM culture medium with 10% FBS at 37 °C for 24 h.For imaging ClO − and N 2 H 4 , the HeLa cells were washed with PBS three times to remove culture medium and then pre-treated with PTMQ (10 μM) for 30 min.Afterwards, the cells-containing dishes were washed there times with PBS, and mounted on the stage of confocal laser-scanning microscope.Before fluorescence imaging, the HeLa cells were further coincubated with different concentrations of ClO − and N 2 H 4 for another 30 min, respectively.

Sensing performance of PTMQ for ClO − and N 2 H 4
The UV-Vis and fluorescent spectra titration of PTMQ with HClO and N 2 H 4 were measured, respectively (Figs. 1 and 2).As shown in Fig. 1, PTMQ itself is non-fluorescence in DMSO/PBS solution due to the strong intramolecular charge transfer (ICT) effect from sulfur (\ \S\ \) and nitrogen (\ \N\ \) atoms in phenothiazine to quinolinium [30].Upon the addition of an increasing amount of ClO − , the absorption band of PTMQ at 518 nm gradually decreased with the increase of a new band at 465 nm, and simultaneously the color of the PTMQ solution turned from deep purple to light pink.The isosbestic point in the absorption titration spectra implied that PTMQ has transformed to a new compound after reaction with ClO − .Meanwhile, a fluorescence band at 577 nm was lighted-up, which progressively rose up with the increasing amount of ClO − from 0 to 500 μM (Fig. 1B).Notably, a good linear correlation between fluorescence intensity at 577 nm (F 577 , R 2 = 0.9912) and concentration of ClO − (0-500 μM) was observed (Fig. 1C), and the detection limit was calculated as 58 nM (S/N = 3).Moreover, the time-dependent fluorescence profile showed that F 577 of PTMQ quickly rose up and reached a stable plateau within 12 s (0.2 min) after addition of ClO − (500 μM).This observation suggests that PTMQ could be used as a rapid-response probe for ClO − detection in aqueous solution.
The spectral responses of PTMQ towards N 2 H 4 were shown in Fig. 2. With the addition of increasing amount of N 2 H 4 , the absorption band of PTMQ at 518 nm gradually decreased along with the increase of a new band at 285 nm, and the color of PTMQ solution changed from deep purple to colorless in synchrony.Notably, a green fluorescence band emerged at 500 nm, which were becoming much stonger with the increase of N 2 H 4 amount from 0 to 900 μM (Fig. 2B).Likewise, a perfect linear correlation between fluorescence intensity at 500 nm (F 500 , R 2 = 0.9930) and N 2 H 4 concentration (0-900 μM) was observed (Fig. 2C), and the detection limit was calculated as 89 nM (S/N = 3).From the time-dependent fluorescence enhancement profile, it could be observed that after the addition of N 2 H 4 (900 μM), F 500 of PTMQ gradually enhanced and reached a stable plateau within 24 min, implying that PTMQ took a relatively slow chemical reaction with N 2 H 4 .Therefore, it provides a temporal and spectral discriminative manner to determine N 2 H 4 and ClO − .

Effect of pH on the sensing performance
As an important environmental factor, pH value usually has a large impact on the chemical stability and sensing capability of fluorescence probes [31].Herein, the pH-dependent fluorescence response of PTMQ (10 μM) to ClO − (500 μM) and N 2 H 4 (900 μM) were investigated, respectively.As depicted in Fig. 3A, the fluorescence intensity at 577 nm (F 577 ) of PTMQ almost maintained constant within the pH range of 3.0-9.5.Upon the addition of ClO − (500 μM) to PTMQ solution, an obvious fluorescence enhancement was observed at pH 5.0-8.5.Whereas, the fluorescence intensity at 500 nm (F 500 ) of PTMQ also kept constant within the pH range of 3.0-10.0,as shown in Fig. 3B.The addition of N 2 H 4 (900 μM) also led to a sharp fluorescence enhancement at pH 6.0-9.5.Therefore, PTMQ itself owns good pH stability and remarkable fluorescence response to ClO − and N 2 H 4 at pH 7.4, which is suitable for intracellular bioimaging applications.

The selectivity
For practical detection, a favorable fluorescent probe should have high selectivity to the analyte.Herein, we tested the selectivity of PTMQ (10 μM) to ClO − (500 μM), N 2 H 4 (900 μM) and various species  ), metal cations (Zn 2+ , Al 3+ , Ba 2+ , Ca 2+ , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Na + , Ni 2+ ) as well as biothiols (Cys and GSH).As shown in Fig. 4A, negligible fluorescence and color changes of PTMQ were observed in the presence of various species except ClO − .In contrast, the addition of ClO − brought an evident bathochromic shift from purple to light pink with an obvious fluorescence enhancement at 577 nm.Likewise, PTMQ (10 μM) also demonstrated remarkable green fluorescence "turn-on" response to N 2 H 4 (100 μM) along with color change from purple to colorless (Fig. 3B).The results indicated that probe PTMQ possesses excellent selectivity to ClO − and N 2 H 4 , which might be employed as a potential tool for real sample detection.

Working mechanism
To further understand the working mechanism, we made great efforts to isolate the major products generated from the reactions of PTMQ with ClO − and N 2 H 4 , respectively.Unfortunately, we could not obtain the pure products for NMR analysis due to their strong polarity and some complex fragments.So, we can only measure the HRMS spectra of main products.After PTMQ was treated with ClO − , the HRMS spectra of PTMQ (Fig. S8) showed a dominant peak located at m/z value of 465.1649 (calcd: 465.1612), which was corresponding to [PTMQ-O + ].It was proposed that the electron-donating sulfur atom (\ \S\ \) in phenothiazine was converted into electron-withdrawing sulfoxide (-S=O) group after reaction with ClO − [30], which blocked the intramolecular charge transfer effect (ICT), and thus leading to a significant red fluorescence enhancement.For the sensing mechanism of N 2 H 4 , the HRMS spectra (Figs.S9 and S10) showed two main peaks at m/z values of 310.1026 and 172.1146 after reaction with N 2 H 4 , which were identified as PT-NHNH 2 (calcd: 310.1051) and MQ (calcd: 172.1027), respectively.These observations indicated that the polarized C_C bridge of PTMQ could be disrupted by nucleophilic N 2 H 4 to form PT-NHNH 2 [17], which gave rise to green fluorescence emission.Based on these observations, the proposed working mechanisms of PTMQ for HClO/ClO − and N 2 H 4 were depicted in Scheme 2.

Cell imaging
In view of the aforementioned excellent sensing properties, PTMQ was utilized to separately image HClO and N 2 H 4 in human cervical cancer (HeLa) cells [32,33].Firstly, the HeLa cells were pre-incubated with PTMQ (10 μM) at 37 °C for 30 min, and the fluorescence images were recorded by laser confocal scanning microscopy.As illustrated in Fig. 5A and B, for the blank cells, non-fluorescence could be observed in red and green channels.Then, these HeLa cells were co-incubated with different amounts of ClO − (0, 80 and 200 μM) and N 2 H 4 (0, 300, and 900 μM) for 30 min, and their fluorescence images were recorded.The cells after incubation with 80 μM of ClO − displayed weak red fluorescence emission, which became much brighter with the increase of ClO − concentration from 80 μM to 200 μM.By contrast, the cells showed clear green fluorescence at N 2 H 4 concentration of 300 μM and remarkable green fluorescence emission when the concentration rose up to 900 μM.These observations demonstrated that PTMQ could serve as an efficient fluorescent probe for separately imaging of exogenous ClO − and N 2 H 4 in living cells.Moreover, it can be observed that the intracellular green/red fluorescence of PTMQ induced by ClO − and N 2 H 4 mainly localized in the cytoplasm within 30 min, suggesting that ClO − and N 2 H 4 can quickly penetrate and diffuse inside the HeLa cell, which may eventually cause oxidative-injury and/or hepatic steatosis [34].
The dual-analytes responsive properties, high contrast ratio, and low background interference, enable PTMQ to serve as a promising tool for discriminatively bioimaging and monitoring ClO − and N 2 H 4 .

Conclusion
In conclusion, we have designed and synthesized a dual-analytes responsive fluorescent probe PTMQ for distinguishing detection of ClO − and N 2 H 4 in aqueous solution.PTMQ exhibited a colorimetric and ratiometric fluorescence response to N 2 H 4 with good selectivity and a low detection limit (89 nM).Meanwhile, PTMQ displayed red fluorescence lighting-up response to ClO − with good selectivity, rapid response (<0.2 min) and a low detection limit (58 nM).Moreover, PTMQ showed good biocompatibility and could be applied for in-situ imaging of ClO − and N 2 H 4 in living HeLa cells.This work provides an efficient way to create dual-analytes responsive fluorescent probe with the advantages of visual detection, good selectivity, as well as in situ discriminative bioimaging of two analytes in cancer cells.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contents lists available at ScienceDirect Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy j o u r n a l h o m e p a g e : w w w .e l s e v i e r .c o m / l o c a t e / s a a response of PTMQ to ClO − and N 2 H 4 , respectively.Moreover, PTMQ was used to in-situ image ClO − and N 2 H 4 in living human cervical cancer HeLa cells.