The Novel Fluorescent Probe Toward Yttrium(III) and its Bioimaging

In this paper, the novel fluorescence probe XP based on Schiff-base was designed, synthesized and characterized, which could detect Y3+selectively and sensitively. The recognition mechanism of XP toward Y3+ was studied by Job's plot and HRMS. It was investigated that stoichiometric ratio of the probe XP conjugated with Y3+ was 1:2. And the detection limit was calculated as 0.30 μM. In addition, Y3+ was recognized by the test paper made from XP. And the probe XP could detect Y3+ selectively in Caenorhabditis elegans and the main organs of mice. Thus, XP was considered to have some potential for application in bioimaging.


Introduction
Rare earth ions were significant in our many kinds of domains [1]. Yttrium (Y 3+ ) was one of the rare earth ions [2,3], and it was regarded as the relatively disperse element [4]. During the past two decades, it was used in lots of aspects [5], such as targeted radiotherapy [6], catalysis [7], fluorescent materials [8], model surface for investigating DNA hybridization [9], and the synthesis of nanometre materials [10,11]. However, due to more and more application of Y 3+ , it produced some radioactive waste which leaded to environment contamination [12], and Y 3+ was the most poisonous element in itself in organisms [13]. So, it was essential to seek some rapid, sensitive and convenient method to detect Y 3+ .
Nowadays, compared to traditional detecting means [14,15], the fluorescence probe was got closely concerned and attention by more and more researchers because of its sensitivity, low cost, real-time response and simplicity [16][17][18][19][20]. However, few literature reported that Y 3+ was distinguished from lots of rare earth ions [21][22][23].The probes involved in these reports still had some shortcomings, such as complex structure, expensive raw materials, inconvenient synthesis and insufficient research on application potential. Therefore, it was urgent to explore a kind of fluorescent probe which was easy to synthesize and efficient to recognize Y 3+ .
For this context, we reported a novel Schiff-base fluorescent probe XP which was designed, synthesized and characterized. Due to containing imine bond, it was offered possibility to combine with metal ions [24,25], showing an excellent recognition and sensitivity ability towards Y 3+ with "turn-on" fluorescence phenomenon. Compared with previous fluorescence probe, its chemical structure was so simple that the synthesis of XP was easy. The mechanism of XP toward Y 3+ was studied by Job's plot, FT-IR spectrum and HRMS. The detection limit was calculated as 0.30 μM. Through the paper strip, it could achieve the purpose of detection. Also, it has been successfully applied to the bioimaging of Caenorhabditis elegans and the main organs of mice. It provided a new molecule for fluorescent probes to detect rare earth ions.

Materials and Experimental Apparatus
All reagents used were of analytical grade and used without further purification. UV spectra was attained through a Shimadzu UV-1601 spectrophotometer, and NMR spectra at 400 MHz was recorded on a Bruker NMR. Luminescence spectrum was performed on a Horiba Fluoromax-4-NIR spectrometer. Biological imaging was performed on a fluorescent inverted microscope and confocal laser scanning microscope. The six-week-old mice were prepared for a tissue imaging experiment. The tissue imaging experiments of mice were performed by the Research Center of Antiaging Chinese Herbal Medicine of Anhui Province (Fuyang, China) and were treated with standard procedure.

Synthesis of XP
XP was synthesized from 2-hydroxy-1-naphthaldehyde (A) (Fig. 1). Benzoic acid (50 mmol) and thiosemicarbazide (50 mmol) were dissolved in phosphoryl trichloride (13 mL), refluxing and stirring for 2.5 h. And then cool it down to room temperature. Next, add deionized water (75 ml) drop by drop and heat to 110 ℃ for 4 h. Then pH of the product was regulated to 8 by 40% NaOH and precipitation was produced. After recrystallization, we got intermediate product (B) [26]. B was mixed with A in ethanol, and refluxed at 80 ℃ for 8 h. After the reaction was completed, the solvent was vaporized under vacuum and the crude product was purified by recrystallized to get orange substance with 75% yield. 1 S1). 13

Bio-imaging of Caenorhabditis Elegans
Caenorhabditis elegans were incubated with solution of XP and XP + Y 3+ for 10 min individually, and one group of elegans was blank group. Fluorescence bio-imaging was observed under the fluorescence microscope.
Bio-imaging of Organs of Mice Two mice were injected XP and XP + Y 3+ solution respectively, one mouse was as the blank group, and the mice were fed for 6 h. The imaging effects were observed under the fluorescence microscope. The animal experiments were carried with standard procedure, and were performed by Research Center of Anti-aging Chinese Herbal Medicine of Anhui Province (Fuyang, China).

Spectral Analysis of XP
The recognition of XP (10 μM) was analysed by rare earth ions (nitrates of Pr 3+ , Yb 3+ , Eu 3+ , La 3+ , Y 3+ , Pd 2+ , Nd 3+ , Fig. 1 The synthetic routes of probe XP Ce 3+ , Sr 2+ ) in CH 3 CN. As Fig. 2 showed, under 365 nm UV light, XP solution displayed no fluorescence in CH 3 CN. After it interacted with Y 3+ , the solution presented a kind of green fluorescence. Whereas, other rare earth ions failed to produce the phenomenon. Then, there was an obvious emission signal at 525 nm upon the addition with Y 3+ (Fig. 3a), and an obvious nearly twofold fluorescence intensity was enhanced compared to XP with other rare earth ions. Interestingly, the noticeable fluorescence change wasn't observed on other rare earth ions. This suggested that the probe XP had the potential to specifically identify Y 3+ from some common rare earth ions. In addition, some metal ions were also evaluated (Fig. S4). The results showed that the probe XP showed a weak response to Zn 2+ . However, this did not affect the efficient recognition of Y 3+ by the probe XP, since the fluorescence intensity of the system in the presence of Y 3+ was more than twice that of Zn 2+ (Fig. S4). To further investigate the antiinterference ability of the probe XP against other rare earth ions, competitive experiment was operated. XP + Y 3+ was investigated with other rare earth ions (Pr 3+ , Yb 3+ , Eu 3+ , La 3+ , Pd 2+ , Nd 3+ , Ce 3+ , Sr 2+ ) in the following Fig. 3b. The results showed that the coexistence of rare metal ions had little effect on the fluorescence intensity of combination of XP + Y 3+ . Hence, probe XP had ability to recognize Y 3+ under coexistence other rare earth ions. Moreover, we compared with other probes toward Y 3+ (Table S1), and the results also showed that probe XP had some outstanding advantages.

Sensing Mechanism
Under optimized conditions, chemical titration was measured to study the mechanism between XP and Y 3+ . With the concentration changed from 1 μM to 21 μM, the intensity was gradually enhanced. The fluorescence intensity reached the maximum with 20 μM of Y 3+ at 525 nm (Fig. 4a). In addition, by formula of detection limit (LOD = 3δ/K), the LOD was 0.30 μM (Fig. S5). As seen in the Fig. S6, the concentration of complexation of XP + Y 3+ was kept at 33.3 μM, and the maximum fluorescence intensity occurred at near 0.7. Therefore, the results were speculated that the binding ratio of XP on Y 3+ was 1:2. Then, the interaction was studied using FT-IR and HRMS. HRMS results showed an ion peak at an m/z 508.8864 which matched [XP + 2Y 3+ + H + ] + well (calcd. 508.8863) (Fig. S7). The FT-IR was consistent with Job's plot and HRMS results. It was obvious that the peak at 3447.93 cm −1 disappeared, and the peak at 1603.76 cm −1 was changed to 1584.95 cm −1 , so it can be inferred that Y 3+ ion combine with -OH and -C = Nrespectively (Fig. S8). According to the above data, the reaction mechanism was demonstrated as we proposed (Scheme 1). Fig. 2 The photograph of fluorescent probe XP (10 μM) and after adding of rare earth ions (Pr 3+ , Yb 3+ , Eu 3+ , La 3+ ,Y 3+ , Pd 2+ , Nd 3+ , Ce 3+ , Sr 2+ ) in CH 3 CN under the 365 nm UV light

Applications of Paper Strip
We prepared that the probe XP was put into test paper to investigate its potential in practical application. Under the UV lamp, the fluorescence of paper strips got changed to green after adding Y 3+ . However, other rare earth ions didn't have any marked transform. Thus, this paper strip can be effective, real-time to monitor Y 3+ conveniently (Fig. 5).

Bio-imaging of Probe XP and Y 3+ in Caenorhabditis Elegans and Mice Organs
Firstly, the cytotoxicity was studied in HeLa cell. It was demonstrated that the cytotoxicity of the probe XP and XP + Y 3+ were low at low concentrations (Fig. S9). It offered the possibility to perform fluorescence imaging in organisms. As observed in Fig. 6, the blank group and containing XP group of elegans had no fluorescence, however, the fluorescence of complexation of probe XP with Y 3+ was detected as a green fluorescence.
Further step study in endogenous biological aspects, the recognition ability on probe XP to Y 3+ was measured on the organs of mice (liver, kidney and heart). It was obvious to show that a green fluorescence occurred in three organs of mice through probe XP and Y 3+ were injected to body of mice. Nevertheless, the probe XP was introduced to mice in vivo, which emitted no fluorescence. Hence, the probe XP had biological imaging capability to detect Y 3+ in live system, and had potential in biological applications (Fig. 7).

Conclusion
In conclusion, the fluorescence probe XP was designed, synthesized and characterized, which had good binding ability for Y 3+ . The detection limit was as low as 0.30 μM. The 1:2 stoichiometric ratio of XP-Y 3+ was confirmed by many methods. In biological relevant field, it was researched that probe XP had good bio-imaging properties in Caenorhabditis elegans and organs of mice. Thus, probe XP had practice potential in vivo and gave a good example for the detection of Y 3+ from some common rare earth ions.