2-benzothiazoleacetonitrile based two-photon fluorescent probe for hydrazine and its bio-imaging and environmental applications

A novel turn-on two-photon fluorescent probe NS-N2H4 was developed with the 2-benzothiazoleacetonitrile as a new recognition site for the detection of hydrazine (N2H4). The two-photon probe exhibited favorable properties including high selectivity, low cytotoxicity and almost 16-fold fluorescence enhancement in the presence of N2H4 in solution. The probe could be used to image hydrazine in the living cells. Notably, we also used the two-photon fluorescent probe to image hydrazine in the tissue imaging for the first time. Furthermore, by the way of probe-loaded TLC plate, we further monitored vapor of hydrazine. Therefore, the novel two-photon probe is expected to be employed to detect N2H4 in biosamples and environmental pollution and the new recognition site will be widely applied to construct fluorescent probes for the detection of N2H4.

Hydrazine (N 2 H 4 ) has been widely employed in space system as rocket propellant due to its special chemical properties including flammability and explosion 1 . According to its basic and reductive properties, hydrazine has been used as catalyst, corrosion inhibitor, and reducing agent in pharmaceutical, agricultural, and applied chemical industries [2][3][4] . However, it is also regarded as an important industrial pollutant to humans and animals with high toxicity, which could cause the lungs, livers, and kidneys cancerous 5 . Thus, the concentration of N 2 H 4 must be controlled as low as 10 ppb 6 . Therefore, it is highly significant to develop powerful means for the tracking and detection of N 2 H 4 in living systems with high sensitivity and good selectivity.
There are some analytical methods for the detection of N 2 H 4 , which were exploited in the previous work, such as including chromatography-mass spectrometry, titrimetry and electro-chemical methods 7,8 . However, sophisticated instrumentation and highly personal operating techniques must be needed in these processes, which are complex and time-consuming. In the past few decades, organic fluorescent probes, which were regarded as the most powerful monitoring tools, have become an important tool used in biological studies with excellent merits including high sensitivity, good selectivity and real-time detection [9][10][11][12] .
Very recently, a number of fluorescent probes for monitoring N 2 H 4 in living biosystem have been reported  , most of which were reported by deprotection of the leaving group for the detection of hydrazine [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] . Also, only few examples were developed by the cleavage of carbon-carbon double bond [28][29][30] . Besides, some fluorescent probes were used for the detection of N 2 H 4 by the way of open ring, closed ring and effect of ESIPT after reacting with N 2 H 4 [31][32][33][34] . Hence, it is very crucial to develop a new recognition site for the detection of N 2 H 4 . Furthermore, all the previous probes were excited by one-photon wavelengths leading to photobleaching of fluorescent dyes and damage to living cells and tissues. Although the two-photon confocal microscopes are relatively not common, there are significant merits of two-photon microscopy (TPM) with long excitation wavelengths such as three-dimensional detection of living tissues, depressed the photodamage to biological samples, increased penetration ability of tissue and reduced fluorescent interference of background. Therefore, it is very important and necessary to construct two-photon fluorescent probe, which could be suitable for imaging N 2 H 4 specifically in living cells and tissues.
In this report, we have constructed a novel two-photon fluorescent probe NS-N 2 H 4 for the detection of N 2 H 4 with 2-benzothiazoleacetonitrile as a new recognition site. (Fig. 1 the first tissue imaging confirmed that the probe NS-N 2 H 4 can be used to monitor the level of N 2 H 4 in living biosystem. Furthermore, the probe NS-N 2 H 4 could monitor vapor of hydrazine by the way of probe-loaded TLC plate. Therefore, the two-photon probe is expected to be employed to detect N 2 H 4 in biosamples and environmental pollution.

Results and Discussion
Design and synthesis. The platform of 6-hydroxy-2-naphthaldehyde was chosen as the fluorescence reporting group due to two-photon properties and easy modification. By engineering a new recognition moiety of 2-benzothiazoleacetonitrile to the fluorescent platform, we designed and synthesized the two-photon probe NS-N 2 H 4 , which was outlined in Fig. 2 by condensation reaction in one step easily with good yield. The structure of target compound NS-N 2 H 4 was fully characterized by 1 H NMR, 13 C NMR and HRMS.

Fluorescent properties of NS-N 2 H 4 .
With the two-photon probe NS-N 2 H 4 in hand, its optical properties were measured in the absence or presence of N 2 H 4 including absorption (Fig. S1) and fluorescence spectroscopy. The probe NS-N 2 H 4 showed almost no fluorescence with excitation at 360 nm (Fig. 3). In presence of N 2 H 4 , the probe NS-N 2 H 4 exhibited strong emission at 448 nm in PBS-DMSO (v/v = 2/1, pH = 7.4) at ambient temperature. That is to say, PBS-DMSO (v/v = 2/1, pH = 7.4) was regarded as the suitable solvent for the fluorescence experiment. With the time extended, the fluorescence intensity was increased gradually (Fig. 3). Notably, a large fluorescence enhancement (up to 16-fold) was shown. The two-photon probe showed relatively high sensitivity in presence of N 2 H 4 under the experimental conditions, indicating that the probe could be used as a practical tool for the detection of N 2 H 4 . In addition, the probe NS-N 2 H 4 is also stable under irradiation depicted in the Fig. S2.

Mechanism.
To get insight into the proposed sensing process, we studied the reaction of NS-N 2 H 4 with N 2 H 4 by mass spectrometry. When the probe NS-N 2 H 4 (20 μM) was treated with N 2 H 4 (400 μM) in pH 7.4 PBS/DMSO (v/v = 2/1) at room temperature, a significant peak at m/z 187.0875 corresponding to the product NS-N 2 H 4 -adduct appears in the ESI-MS spectrum (Fig. S4), in good agreement with the proposed sensing mechanism ( Fig. 1).

Effect of pH.
We then decided to examine the effect of pH on the fluorescence response of NS-N 2 H 4 to N 2 H 4 .
As shown in Fig. 4, the emissions intensities of NS-N 2 H 4 are quite low and do not change significantly over wide   Scientific RepoRts | 7: 1530 | DOI:10.1038/s41598-017-01656-w ranges of pH from 2.0-9.5, indicating that the free probe was stable in the wide pH range. Upon treated NS-N 2 H 4 with N 2 H 4 , we found that the pH value of solution has a great influence on the probe NS-N 2 H 4 response to N 2 H 4 . With the increase of pH from 7.0 to 9.5, an enhancement trend is observed in NS-N 2 H 4 fluorescence intensity of response to N 2 H 4 , which covers well the physiological pH range of mitochondria (about pH 7.99), indicating that the free probe is suitable for detecting N 2 H 4 in living cells and tissues.
Response rate and selectivity. The time courses of the fluorescence intensities of NS-N 2 H 4 (10 µM) in the presence of N 2 H 4 (20 equiv) in pH 7.4 PBS/DMSO (v/v = 2/1) was measured in Fig. 5. Notably, a gradual increase in fluorescence intensity was observed in the presence of N 2 H 4 in 240 min at ambient temperature. However, the fluorescence intensity increased rapidly at 37 °C (Fig. S3). The fact is that the probe NS-N 2 H 4 could be fit for the detection of N 2 H 4 in real time. The high selectivity to the target molecule over other potentially competing molecules is another important property for a bioimaging probe with potential application in the biosystem. Therefore, we had performed some research on the selectivity of the free probe NS-N 2 H 4 with various relevant analytes including anions, metal ions, reducing agents, small molecule thiols, and N 2 H 4 to investigate the selectivity. As shown in Fig. 6, When other analytes such as Al 3+ , Ca 2+ , Co 2+ , Cu + , Cu 2+ , Mg 2+ , Zn 2+ , S 2− , SO 3 2− , Cys, Cl − were treated with NS-N 2 H 4 , the fluorescence intensity was almost unchanged compared with a strong fluorescent response when treated with N 2 H 4 . These results suggest that the probe NS-N 2 H 4 is highly selective for N 2 H 4 over other tested species.
Application in vapor gas detection. Encouraged by the above excellent properties of the probe NS-N 2 H 4 , we evaluated its potential utility for the detection of hydrazine in real samples. At the beginning, TLC plates were soaked in the solution of NS-N 2 H 4 (0.1 mM, in DMSO). After dried, the NS-N 2 H 4 probe-loaded TLC plates were used to detect gaseous hydrazine, which can further discriminate hydrazine aqueous solutions with different concentrations (Fig. 7). When exposed to the vapor of hydrazine for 10 min, distinctive fluorescence color changes of NS-N 2 H 4 -loaded TLC plates were observed (Fig. 7b-f), which were highly dependent on the concentration of hydrazine in aqueous solution and easy to be distinguished by the naked eyes. However, no visible change was   Fig. 7a). Therefore, these results demonstrate that the probe

NS-N 2 H 4 is suitable for the instant visualization of trace amounts of hydrazine in environmental samples
Bioimaging in living cells. The above measurements indicate that the two-photon fluorescent probe has good properties including sensing appropriately at physiological pH, a very large turn-on signal, in particular a new recognition site, good selectivity. Thus, the probe NS-N 2 H 4 seems to be fit for the detection of N 2 H 4 in real biosamples. We evaluated NS-N 2 H 4 imaging assays in live cells, and fluorescence imaging experiments were carried out in living cells (HeLa cells) on confocal laser scanning microscopy.
The cytotoxicity of NS-N 2 H 4 was examined toward Hela cells by a MTT assay (see Supplementary Fig. S5). The results have proved to be that the probe NS-N 2 H 4 at the low concentrations has no marked cytotoxicity to the cells after a long period (>90% HeLa cells survived after 24 h with NS-N 2 H 4 (30.0 µM) incubation). Therefore, the probe NS-N 2 H 4 is suitable for imaging N 2 H 4 in living cells due to the low cytotoxicity.
After established the excellent sensing performance and the low cytotoxicity of the probe NS-N 2 H 4 , we examine whether the probe could be functional in living cells. The utility of NS-N 2 H 4 for fluorescence imaging of N 2 H 4 in living cells was investigated (Fig. 8). When HeLa cells were incubated with NS-N 2 H 4 for 30 min, no detectable fluorescence was observed. However, when the cells were pre-treated with NS-N 2 H 4 for 30 min and then incubated with N 2 H 4 (10 equiv) solution for another 30 min, the strong fluorescence was shown in the blue channel (Fig. 8e) at the same test conditions, confirming that the probe possess good membrane permeability and could image N 2 H 4 in cellular environment.
Bioimaging in living tissues. Encouraged by the above ideal results of the probe in the blue channel for monitoring N 2 H 4 and the advantages of two-photon fluorescence microscopy, we decided to further operate experiment for the detection of N 2 H 4 in living tissues by two-photon fluorescence microscopy. The living tissues slices of the fresh rat liver were prepared with thickness at 400 μm, which were measured by two-photon fluorescence microscopy. At the beginning, tissue slices incubated with only the probe NS-N 2 H 4 (20.0 μM) for 30 min at 37 °C in PBS exhibit no fluorescence at the emission window of 0-75 nm (Fig. S6). When tissue slices were incubated with NS-N 2 H 4 (20.0 μM) for 30 min, and then treated with N 2 H 4 (20 equiv) for another 30 min, significant fluorescence emerged up to 75 μm depth of living tissues by the way of two-photon fluorescence microscopy, which has exhibited its two-photon fluorescence properties (Fig. 9).

Conclusions
In conclusion, we have developed a turn-on two-photon fluorescent probe with the 2-benzothiazoleacetonitrile as a new recognition site for the detection of hydrazine N 2 H 4 . Desirable properties including good selectivity and low cytotoxicity are emerged. The probe NS-N 2 H 4 could be used to image hydrazine in living cells as well   Tissue imaging. The Kunming mice were purchased from Shandong University Laboratory Animal Center (Jinan, China). All procedures for this study were approved by the Animal Ethical Experimentation Committee of Shandong University according to the requirements of the National Act on the use of experimental animals (China).The fresh mouse liver slices were obtained from the liver of 14-day-old mouse. The living liver slices were gained with 400 micron thickness using a vibrating-blade microtome in 25 mM PBS (pH 7.4). The living liver slices were pre-treated with NS-N 2 H 4 (20 μM) for 30 min. The slices were washed three times by PBS buffer and imaged. To obtain the two-photon fluorescence images of the tissues incubated with both the probe and anlysis sample (N 2 H 4 ), the slices were pre-treated with NS-N 2 H 4 (20 μM) for 30 min before the N 2 H 4 was added. Following this incubation for another 30 min at 37 °C, the slices were washed three times by PBS buffer and imaged. The two-photon fluorescence emission was collected at between 420 and 495 nm upon excitation at 800 nm with a femtosecond laser.
Synthesis of the probe NS-N 2 H 4 . A mixture of 6-hydroxy-2-naphthaldehyde (0.5 mmol, 100.0 mg, 1.0 equiv) and benzothiazole-2-acetonitrile (0.55 mmol, 58.9 mg, 1.1 equiv) were dissolved in EtOH (5.0 mL). The piperidine (0.55 mmol, 46.8 mg, 1.1 equiv) was added under N 2 . After stirred at room temperature for 8 h, the reaction mixture was adjusted to distilled water (2.0 mL), and then extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride, dried over Na 2 SO 4 , filtered, and concentrated under vacuum, and the product was obtained by silica column chromatography to give the probe NS-N 2 H 4 in the yield of 83%. 1