Introduction

Hydrazine is widely used as an important industrial material in chemical, pharmaceutical, and agricultural areas due to its vital roles [1–8]. And it is well-known as high-energy fuel in rocket propulsion and missile systems owning to its improved detonable properties. However, hydrazine is extremely toxic and can be easily absorbed by oral, dermal and inhalation exposure routes [9–11]. It should be noted that chronic exposure to high concentration hydrazine may damage various organs and destroy the central nervous system [4, 12, 13]. As a result, hydrazine has been classified as a potential human carcinogen substance by the U.S. Environmental Protection Agency (EPA) and World Health Organization (WHO) with allow threshold limit value (TLV) of 10 ppb [14, 15]. Therefore, it is urgently needed to develop effective analytical techniques to detect trace amount of hydrazine with satisfactory selectivity and sensitivity.

Various hydrazine detection methods, such as chemiluminescence, [16] electrochemical analysis [17, 18] and chromatography [19] have been developed for hydrazine detection. Unfortunately, they are always expensive, time-consuming and complicated, in addition, they are not suitable for real-time and on-site analysis. The fluorescent spectroscopic technique has received increasing attention due to its high sensitivity and specificity, non-invasiveness, and good compatibility for biosamples [20–25]. Till now, a number of fluorescent probes for hydrazine have been developed [26–50]. However, most of them need long response time and few of them work under neutral conditions or the unsatisfactory detection limit. So far, chemical small-molecular fluorescent probes for hydrazine are still very limited. Thus, it has been still a great challenge to develop facile and reliable small-molecule fluorescent probers for the rapid and effective determination of hydrazine both qualitatively and quantitatively.

The reported hydrazine detection probe chose malononitrile group [26–32], acetate group [33–40], 4-bromobutyrate masking unit [41–43], trifluoroacetylacetone group [44, 45], phthalimide group [46–48] or aldehyde group [49, 50] as the sensing part. In this work, we chose р-nitrobenzoic acid as the sensing part. We envision that the strong electron-withdrawing group (−NO2) will contribute to the deprotection, thus the response time or detection limit will be better. As we expected, this probe can distinguish hydrazine over other biologically relevant anions and cations in about 2 min in PBS-DMSO (10 mM, pH = 7.4, 1:4, v/v) solution at room temperature with a detection limit of 4.6 × 10−10 M. To the best of our knowledge, the detection limit is lower than most of the reported [Table S1]. It also has the shortest response time except Xu’s reported [46, 48]. Herein, we reported its simple synthesis, optical properties and application for imaging of hydrazine in living MIA PaCa-2 cells.

Experimental

Apparatus Regents and Chemical

All chemicals and reagents were used as received from commercial sources without further purification. Deionized water was used throughout all experiment. Hydrazine and other ions stock solutions (10.0 mM) were prepared in water, while stock solution of probe FN (1.0 mM) was prepared in DMSO. The stock solution was diluted to obtain the final concentration of the probe with the solution of PBS-DMSO (10 mM, pH = 7.4, 1:4, v/v) 1H and 13C NMR spectra were carried out on a Bruker DTX-400 spectrometer using TMS as internal reference. ESI mass spectra were performed on an HPLC Q-Tof HR-MS spectrometer. UV-vis spectra were measured on an Agilent 8453 spectrophotometer. Fluorescence spectra measurements were performed on a HITACHI F-7000 fluorescence spectrophotometer. The melting points were determined by an X-4 microscopic melting apparatus with a digital thermometer (Shanghai, China). The pH was measured with a Model pHs-3C meter (Shanghai, China).

Synthesis of Probe FN

р-nitrobenzoic acid (500 mg, 3 mmol), EDC (570 mg, 3 mmol) and DMAP (120 mg, 1 mmol) were combined and dissolved in DCM (25 mL). The reaction mixture was stirred for 10 min at room temperature followed by the addition of fluorescein (330 mg, 1 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the crude product was purified by column chromatography on silica gel using CH2Cl2 as eluent to give FN as a white solid (79 % yield). mp 178–180 °C; 1H NMR (400 MHz, DMSO-d6, ppm): δ 8.40 (dd, J = 8.4 Hz, 8 H), 8.10 (d, J = 7.4 Hz, 1 H), 7.78–7.88 (m, 2 H), 7.57 (s, 2 H), 7.46 (d, J = 7.4 Hz, 1 H), 7.20 (d, J = 8.3 Hz, 2 H), 7.01 (d, J = 8.6 Hz, 2 H); 13C NMR (100 MHz, CDCl3, ppm): 169.03, 162.81, 152.76, 151.84, 151.64, 151.08, 135.48, 134.34, 131.44, 130.28, 129.33, 126.09, 125.42, 124.05, 123.84, 117.72, 117.14, 110.47, 81.39. HR-MS: m/z, calcd for C34H18N2O11 [M + H] + 631.0983, found: 631.0981. ( Supporting Information).

Results and Analysis

Compound FN was synthesized according the reported methods [51]. The structure of title compound FN was well characterized by 1H NMR, 13C NMR and HR-MS (Supporting information, Fig. S1-S3). The results were in good agreement with the structure shown in Scheme 1.

UV-Vis Spectral Responses of FN with Hydrazine

With this probe in hand, we first investigated its absorption spectral changes upon the addition of hydrazine and other analytes in PBS-DMSO (10 mM, pH = 7.4, 1:4, v/v) solution at room temperature. As shown in Fig.1, in the absence of hydrazine, the UV-vis spectra of FN (10 μM) exhibited only very weak absorption owing to the closed spirolactone form of fluorescein. However, upon the addition of hydrazine (10 equiv.), the absorption at 515 nm simultaneously increased resulting from the ring-opened fluorescein. The color of the solution changed from colorless to yellow after the addition of hydrazine. Whereas, no obvious response occurred upon the addition of other analytes including K+, Fe3+, Zn2+, Cu2+, Mg2+, Co2+, Ba2+, Cr3+, Cd2+, Hg2+, Mn2+, Ni2+, Pb2+, F−, Cl−, Br−, I−, CH3COO−, N3 −, NO3 −, HSO3 −, SO3 2−, HCO3 −, H2PO4 − and HPO4 2− under the same test condition. These results demonstrated that FN can be served as a visual probe for hydrazine, which would allow the colorimetric detection of hydrazine directly by the naked-eye.

Fig. 1
figure 1

Absorption spectra of FN (10 μM) with the presence of 10 equiv. various species in PBS/DMSO (10 mM, pH = 7.4, 1: 4, v/v) at room temperature. Insert: the color change observed by the naked eye of the probe with the addition of hydrazine

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Fluorescence Spectral Responses of FN with Hydrazine

Then, we investigated the fluorescence emission spectra of probe FN (Fig. 2). In the absence of hydrazine, the free probe displayed a very weak emission. However, a significant enhancement in fluorescence intensity was observed when hydrazine was added. Other biologically relevant analytes such as K+, Fe3+, Zn2+, Cu2+, Mg2+, Co2+, Ba2+, Cr3+, Cd2+, Hg2+, Mn2+, Ni2+, Pb2+, F−, Cl−, Br−, I−, CH3COO−, N3 −, NO3 −, HSO3 −, SO3 2−, HCO3 −, H2PO4 − and HPO4 2− almost could not lead to any notably changes under the same test condition.

Fig. 2
figure 2

Fluorescence spectra of FN (10 μM) with the presence of 10 equiv. of various species in PBS/DMSO (10 mM, pH = 7.4, 1: 4, v/v) at room temperature (λex = 480 nm. Slits: 2.5 nm). Inset: the fluorescence change of the probe with the addition of hydrazine

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To further demonstrate the ability of FN to detect hydrazine in the presence of other competitive cations and anions, the anti-interference experiment of FN was also conducted. As shown in Fig.3, other competitive analytes did not induce any appreciable fluorescence change at 536 nm. In the presence of these interferents, prober FN still showed the same turn-on fluorescence response to hydrazine as that in the absence of these interferents except that Hg2+ imposed little interference. The above results indicated that FN possessed high selectivity toward hydrazine over other cations and anions.

Fig. 3
figure 3

Black bars represent the solution of FN (10 μM) in the presence of various ions (100 μM). Red bars represent the addition of hydrazine (100 μM) to the above solution, respectively

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The Detection of Hydrazine

Generally, the detection limit is a very important parameter to assess a new probe. We subsequently examined the sensitivity of FN for hydrazine detection by varying concentrations of hydrazine (0–80 μM) (Fig. 4). The continuous addition of hydrazine leads to a gradual increase of emission intensity at about 536 nm. A good linear relationship between fluorescence intensity and hydrazine concentration was found. The detection limit (3σ/slope) [52] of probe FN for the determination of hydrazine was found to be 4.6 × 10−10 M, which is far below the TLV (10 ppb) recommended by EPA. These results suggested that probe FN can detect hydrazine both qualitatively and quantitatively.

Fig. 4
figure 4

[1] The emission spectra of FN (10 μM) upon addition of hydrazine (0–8 equiv.) in PBS/DMSO (10 mM, pH = 7.4, 1: 4, v/v) at room temperature [2]. The fluorescence intensity of FN at 536 nm versus different concentration of hydrazine (0-8equiv.). (λex = 480 nm. Slits: 2.5 nm)

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Time Dependence

Reaction time is also a vital factor to evaluate the feasibility of a new probe for real time detection. Therefore, the time dependence of the response of FN to hydrazine was evaluated. As shown in Fig. 5, the fluorescence intensity of FN and hydrazine reached a plateau in about 2 min, after which the fluorescence intensity remained almost constant. However, the emission intensity of blank solution (only FN, 10 μM) almost unchanged at the same condition. The time-dependent change plot demonstrates the reaction could complete in about 2 min, which indicates that probe FN has a fast response for hydrazine.

Fig. 5
figure 5

Time-dependent emission changes of probe FN (10 μM) upon addition of hydrazine (10 equiv.) in PBS/DMSO (10 mM, pH = 7.4, 1: 4, v/v)

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Effect of pH

For practical applicability, the proper pH condition of this new probe for hydrazine detection was also evaluated. We investigated the fluorescence properties of probe FN and that probe FN with hydrazine under different pH values, respectively (Fig. S4). It can be seen, probe FN was pH insensitive, and its emission intensity was quite weak from pH 1 to 9. However, the fluorescent sensing toward hydrazine was obviously affected by the change of pH values. Fluorescence signal reach its maximum and keep constant around biologically relevant pH 5 to 9, indicative of its potential for application in biological systems.

Sensing Mechanism

According to the previous reported work [33, 43], we proposed that the strong fluorescence enhancement is attributed to the deprotection of р-nitrobenzoyl group and concurrent generation of fluorescein. Probe FN is colorless and non-fluorescent owning to the closed spirolactone form of fluorescein. After react with hydrazine, the ring-opened fluorescein was released and caused strong fluorescence. In order to verify our speculation, the reaction products of probe FN and hydrazine were subjected to ESI-MS spectrum. A major ion peak was founded at m/z = 331.17 (Fig. S5), corresponding to the resulting fluorescein ([M-H]−), clearly confirmed the proposed mechanism as shown in Scheme 2.

Scheme 1
scheme 1

Synthetic route of target compound

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Scheme 2
scheme 2

The proposed mechanism of the response of FN to hydrazine

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Fluorescence Imaging in Living Cells

Finally, the bioimaging application of probe FN to detect hydrazine in living cells was also investigated (Fig. 6). As controls, MIA PaCa-2 cells were incubated with only FN (10 μM) for 30 min at 37 °C which showed almost no intracellular fluorescence (Fig. 6b). After treatment of N2H4 (50 μM) and then incubated for further 30 min, a significant fluorescence signal was observed and collected via fluorescence microscopy in the green channel (Fig. 6d). Furthermore, a bright field transmission image of cells treated with FN and N2H4 confirmed that these cells were viable throughout the imaging experiments (Fig. 6a and c). Therefore, these results demonstrated that probe FN was cell membrane permeable and capable of fluorescence imaging of hydrazine in biological samples.

Fig. 6
figure 6

Fluorescence images of hydrazine in MIA PaCa-2 cells with 10 μM solution of probe FN for 30 min at 37 °C, Bright-field transmission images (a, c) and fluorescence images (b, d) of MIA PaCa-2 cells incubated with 0 μM, 50 μM of hydrazine for 30 min, respectively

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Conclusions

In conclusion, we have rationally designed and synthesized a fluorescence off-on probe, which can exhibit a good selectivity toward hydrazine over other competitive species, with the detection limit of 4.6 × 10−10 M. The colorimetric and fluorescent response to hydrazine can be conveniently detected even by the naked eye, which provides a facile approach for visual detection of hydrazine. The living cell imaging demonstrated its value of practical application in biological systems.