Introduction

HOCl/OCl− is one of the most important reactive oxygen species (ROS). It is produced by the reaction of chloride ions and hydrogen peroxide catalyzed by myeloperoxidase (MPO) in living organisms and is involved in many physiological and pathological processes in the body (Harrison and Schultz 1976; Kettleet and Winterbourn 1997; Sivaraman et al. 2014; Raja et al. 2017; Ponnuvel et al. 2018; Perumal et al. 2020; Swamy et al. 2020). The change of HOCl concentration is closely related to the functional state of cells. At physiological concentrations, HOCl provides a guarantee for human body to resist pathogen and bacterial invasion through its strong oxidation and bactericidal ability (Chen et al. 2011) However, once the concentration of HOCl is abnormal, it will directly damage organelles and tissues in the body, thus leading to the occurrence of disease. It has been reported that the concentration of ROS in cancer cells is about 10 times higher than that in normal cells (Wang et al. 2021; Antunes and Cadenas 2001; Wang et al. 2022), which may help distinguish cancer cells from normal cells. Therefore, it is still of great significance to track the real-time detection of HOCl in the body.

In recent decades, there have been numerous reports on the detection of HOCl/OCl−, such as mass spectrometry (Peris-Díaz et al. 2021), electroanalysis (Wang et al. 2008), and chemiluminescence. In recent decades, there have been numerous reports on the detection of HOCl/OCl−, such as potentiometric, electroanalytical, and chemiluminescence methods. However, due to the high cost and complicated operation of these methods, more attention has been paid to the effective detection of HOCl by fluorescent probes. The HOCl fluorescent probe design strategy is based on the reaction between HOCl and specific functional groups. At present, the main types reported are oxidation deoxime mechanism (Nguyena et al. 2018), oxidation of sulfur-containing elements (S, Se, Te elements) atom or group mechanism (Kenmoku et al. 2007; Koide et al. 2011; Wu et al. 2017; Yuan et al. 2015; Xu et al. 2015), oxidation of p-methyl phenol or p-methoxyaniline mechanism (Zhou et al. 2012; Sun et al. 2008; Hu et al. 2016, 2014), desulfurization cyclization (Hua et al. 2019), oxidation of carbon–carbon double bond (Zou et al. 2019; Chen et al. 2010), oxidation of deiminomaleonitrile (Zhu et al. 2014; He et al. 2020), etc. (Table 1).

Table 1 Comparisons of this method and other different mechanism for detecting hypochlorous acid/hypochlorite
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In recent years, fluorescent probes have been favored by chemical biologists due to their excellent characteristics such as high sensitivity, good selectivity, short response time, low cost, easy operation, and in situ imaging (Zhu et al. 2018; Chen et al. 2016; Xu et al. 2016). In addition, fluorescent probes can enter a single cell for accurate detection and can realize the detection of active substances or metabolites in organisms, which is of great significance for the development of modern biology. It is well known that outstanding photostability, biological compaction, solubility, reliable molar absorption coefficient, and fluorescence quantum yield are all requisites in the application of developed fluorophores (Dwight and Levin 2016). For this reason, through the continuous attempts and innovations of many researchers, many fluorescent probes have been invented based on 2-(2-hydroxyphenyl) benzothiazole (Zhu et al. 2021a, b), BODIPY (Venkatesan and Wu 2015; Liu et al. 2016; Liu and Wu 2013), coumarin (Duan et al. 2019), fluorescein (Ren et al. 2022), naphthalimide (Feng et al. 2016), naphthalene (Zhang et al. 2020), rhodamine (Xiong et al. 2016; Mao et al. 2019; Yuichiro et al. 2011), 7-nitrobenz-2-oxa-1,3-diazole (NBD) (Jiao et al. 2020), etc. Therefore, it is urgent to synthesize fluorescent probes with simplicity, high sensitivity, good selectivity, low detection limit, and good photostability.

In this paper, a small molecule fluorescent probe, 9-ethyl-3-((2-(4-nitrophenyl) hydrazineylidene)methyl)-9H-carbazole (CZ-NH) with high selectivity for HOCl, was designed by using carbon–nitrogen double bond as the recognition functional group and carbazole with large conjugate system as the fluorophore. CZ-NH showed good quantum yield (Φ = 0.14). When CZ-NH reacts with HOCl, CZ-CHO with strong fluorescence is released, which enhances the fluorescence and achieves the purpose of detection.

Experimental

Materials and chemicals

All other chemicals used in this article were obtained from commercial suppliers and can be used without further purification. The water is deionized. Silica gel for column chromatography was obtained from 200–300 mesh Sinopharm Chemical Reagent Co., LTD.. DMSO-d6 was used as the solvent to record 1H NMR spectrum at 400 MHz and 101 MHz (Bruker DPX) at 13C NMR spectrum. Chemical shifts were reported in ppm with TMS as internal standard. Mass spectra were determined by high-resolution mass spectrometer. Absorption spectra were recorded on a Shimadzu UV-2600 spectrophotometer, and fluorescence spectra were recorded on a Cary Eclipse fluorometer. Cell imaging was recorded on a Leica inverted microscope.

Synthesis of compound CZ-NH

Synthesis of 9-ethyl-9H-carbazole-3-carbaldehyde (CZ-CHO): The solution of DMF (0.13 mL) and 1,2-dichloroethane (3 mL) was put into a round-bottom flask at 0 °C, POCl3 was slowly dropped into the mixture, and then, N-ethyl carbazole dissolved in 1,2-dichloroethane was added to the mixture by drop (Scheme 1). The mixture was heated and stirred at 90 °C for 12 h, and the reaction solution was slowly poured into ice water after the reaction was complete. The products were extracted by ethyl acetate, dried and purified by column chromatography.1H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.74 (d, J = 1.6 Hz, 1H), 8.28 (d, J = 7.8 Hz, 1H), 7.99 (dd, J = 8.6, 1.6 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.53 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 4.47 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 192.31, 143.50, 140.79, 128.71, 127.20, 127.10, 124.49, 122.82, 122.77, 121.33, 120.57, 110.31, 110.00, 37.80, 14.15.

Scheme 1
scheme 1

Design and synthesis of the CZ-NH

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Synthesis of CZ-NH (Zhu et al. 2021a, b): Add CZ-CHO (1 mmol) and p-nitrophenylhydrazine (1.5 mmol) to a round-bottomed flask, dissolve with absolute ethanol, and heat under reflux at 80 °C for 6 h. After the reaction, the excess solvent was removed, and the product was recrystallized from anhydrous ethanol.1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 8.48 (s, 1H), 8.25 (d, J = 5.7 Hz, 2H), 8.15 (d, J = 9.1 Hz, 2H), 7.93 (d, J = 8.5 Hz, 1H), 7.65 (dd, J = 13.7, 8.4 Hz, 2H), 7.49 (t, J = 7.7 Hz, 1H), 7.27–7.16 (m, 3H), 4.46 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 151.34, 143.95, 140.70, 140.45, 138.24, 126.73, 126.61, 126.20, 124.59, 122.88, 122.63, 121.12, 120.08, 119.73, 111.47, 110.03, 109.91, 37.61, 14.24.

Fluorescence experiments

Prepare NaClO stock solutions (1 mM) and other analytes in deionized water. Probe 1 (1 mM) stock solution was prepared in DMSO. Various analyte stock solutions and probe stock solutions were taken into test tubes, and a mixture of DMSO and deionized water (1:1, v/v) containing phosphate-buffered saline (PBS, 20 mM, pH 7.4) was used. Dilute to desired concentration. All measurements were performed at room temperature (25 °C). All spectra were acquired in quartz cuvettes (200 μL). The excitation wavelength was 300 nm, and the excitation and emission slit widths were both 5 nm.

Cell culture and imaging

The cells were placed in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin (80 mg/L), and penicillin (80 units/mL), incubated in a humidified CO2 incubator (37 °C) for 24 h. The cytotoxic effect of CZ-NH on RAW 264.7 cells was determined by standard methylthiazol tetrazolium (MTT) method.

The control group was treated with CZ-NH (10 μM) and washed with PBS buffer for three times. The exogenous and endogenous NaClO groups were pretreated with NaClO (500 μM) or lipopolysaccharide (LPS, 1 mM). The cells were incubated with CZ-NH (10 μM) for 30 min and washed with PBS for three times. Finally, live cells were imaged using a fluorescent inverted microscope.

Results and discussion

The probe mother liquor is composed of DMSO. The fluorescence intensity of the probe solution without NaClO at 370 nm is very weak, while the fluorescence intensity at 370 nm is significantly enhanced after NaClO is added. The results show that NaClO can increase the fluorescence intensity of the probe, because NaClO can oxidatively destroy C=N, while the p-NO2 group is a strong electron-withdrawing group, which makes C=N more unstable. The presence of free CZ-CHO in solution resulted in enhanced fluorescence. It indicates that the CZ-NH can detect OCl− sensitively.

Reaction time is an important indicator to measure whether a probe can be used for monitoring and analysis, so we first studied the specific situation of the reaction time between probe and NaClO. As shown in Fig. 1a, after NaClO was added to the probe buffer solution, the fluorescence intensity of the probe first strengthened with the prolongation of time. When the reaction time reached 20 min, the fluorescence intensity of the probe tended to be stable. The results show that the probe can be used as an effective method for rapid detection of NaClO.

Fig. 1
figure 1

a Effects of time on CZ-NH (10 μM) and its recognition ability for OCl− in the aqueous solution of PBS (10 mM); b Effects of pH on CZ-NH (10 μM) and its recognition ability for OCl− in the aqueous solution of PBS (10 mM). Excitation wavelength was 300 nm, and excitation and emission slit widths were 5 nm. The data represent the fluorescence intensities at 370 nm

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The response of probe to NaClO at different pH is an important factor to determine whether probe can play an effective role. As shown in Fig. 1b, after adding buffer solutions of different pH to the mixture of probe and NaClO, the fluorescence intensity did not change with the change of pH, but tended to a stable state. When pH is 7.4, the fluorescence intensity reached the maximum value. It shows that the probe is suitable for the detection of NaClO in human body.

We also explored the selectivity of the probes for different analytes (including Cu2+, Ni2+, Zn2+, Fe3+, K+, Ca2+, Al3+, Na+, Cys, Hcy, His, Arg, Lys, NO3−, NO2−, Br−, H2PO4−, CH3COO−, ·OH, O2·−, ONOO−, H2O2, 1O2, MnO4−, ClO2−, Cr2O72−), and the fluorescence intensity was significantly enhanced after the addition of NaClO, while the fluorescence intensity did not change significantly when others were added. As shown in Fig. 2, at the wavelength of 370 nm, the fluorescence intensity generated by the addition of NaClO to CZ-NH was significantly enhanced, and the fluorescence intensity increased nearly 500-fold. The results showed that the selectivity of the probe to NaClO was better than that of other components (Table S1).

Fig. 2
figure 2

a Fluorescence intensity of CZ-NH (10 μM) at 370 nm after addition of 10 mM selected ions; b Response values of probe CZ-NH and various analytes (1: Cu2+, 2: Ni2+, 3: Zn2+, 4: Fe3+, 5: K+, 6: Ca2+, 7: Al3+, 8: Na+, 9: Cys, 10: Hcy, 11: His, 12: Arg, 13: Lys, 14: NO3−, 15: NO2−, 16: Br−, 17: H2PO4−, 18: CH3COO−, 19: ·OH, 20: O2·−, 21: ONOO −, 22: H2O2, 23: 1O2, 24: MnO4−, 25: ClO2−, 26: Cr2O72−,27: PBS, 28: OCl.−)

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When the NaClO concentration ranged from 0 to 160 μM, the increase in fluorescence intensity showed a good linear relationship (Fig. 3b). The detection limit of this method is 2.709 μM, and it has good sensitivity for NaClO.

Fig. 3
figure 3

a Fluorescence responses of CZ-NH (10 μM) to different concentrations of OCl− in DMSO-PBS buffer (10 mM, pH 7.4) (V/V = 1:1); b The linear relationship between the fluorescence intensity and the concentration of NaClO. Excitation wavelength was 300 nm, and excitation and emission slit widths were 5 nm. The data represent the fluorescence intensities at 370 nm

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It is known that the conversion of p-nitrophenylhydrazone to aldehyde can be carried out by an oxidizing agent (McMucrry 1968). And hypochlorite has a strong oxidizing property, so the addition of OCl− breaks the C=N in the probe structure, the reactive p-nitrophenylhydrazine group is cleaved and the free fluorophore CZ-CHO is released, resulting in significant fluorescence changes. To further understand the reaction mechanism between CZ-NH and OCl−, the ESI–MS spectrum of CZ-NH in CH3OH treated with OCl− is shown in Supporting Information Fig. 3S. There is a peak at m/z = 224.09, corresponding to [B + H]+ (Cal. 224.10), and m/z = 246.10, corresponding to [B + Na]+ (Cal. 246.10). According to previous research results, the mechanism by which CZ-NH might recognize OCl− is proposed, as shown in Scheme 2.

Scheme 2
scheme 2

Proposed response mechanism of CZ-NH to OCl−

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To further verify the proposed inter-probe mechanism, density functional theory (DFT) calculations were performed. Figure 4 lists the highest and lowest occupied molecular orbitals (HOMOs) for CZ-NH and CZ-CHO. The HOMO of CZ-CHO is mainly distributed on CZ-CHO, and the LUMO is all over the molecule. The large HOMO–LUMO gap (2.75 and 4.07 eV for CZ-NH and CZ-CHO HOMO–LUMO gaps, respectively) shows high stability of CZ-CHO upon addition of OCl− converting CZ-NH to CZ-CHO. The results verify the reaction mechanism, and CZ-NH is highly selective and sensitive to OCl−, which can enhance the fluorescence.

Fig. 4
figure 4

Structure optimization diagram of probe CZ-NH and adding OCl−

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MTT assay was used to evaluate the cytotoxicity of RAW 264.7 cells. The results showed that the cell viability was more than 90% when the probe concentration was below 20.0 μM (Supporting Information Fig. 1S), indicating that CZ-NH had low cytotoxicity. Cells were pretreated with NaClO and LPS for 30 min at 37 °C and then incubated with CZ-NH (10 μM) for another 30 min at 37 °C. A strong blue fluorescence signal appeared in the cytoplasm of the cells (Fig. 5), and the fluorescence of NaClO experimental group was significantly stronger than that of LPS experimental group. When treated with CZ-NH only for 30 min at 37 °C, there was almost no fluorescence signal in the cells (Fig. 5f). These results indicate that CZ-NH can detect NaClO in living cells.

Fig. 5
figure 5

Fluorescence imaging of RAW 264.7 cells. The first column shows cells treated with CZ-NH (10 μM) (a bright field; d blue channel); The second column shows cells treated with NaClO (500 μM) and CZ-NH (10 μM) (b bright field; e blue channel). The third column shows cells treated with LPS (LPS, 1 mM) and CZ-NH (10 μM) (c bright field; f blue channel). Scale bar: 10 μm

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Conclusions

We have successfully designed a novel carbazolyl fluorescent probe, CZ-NH, which can selectively react with NaClO. The addition of OCl− broke the C=N in the probe structure, and the nitro group was a strong electron-withdrawing group, which accelerated the C=N cleavage and released the free fluorophore CZ-CHO, thus producing significant fluorescence changes and realizing its fluorescence detection. The addition of OCl− increased the fluorescence intensity nearly 500 times, low detection limit 2.709 μM, and the probe CZ-NH had low toxicity, good biocompatibility, and could penetrate the cell membrane for intracellular imaging. The good permeability and staining ability further demonstrated the feasibility of CZ-NH to accurately monitor NaClO in biological systems.