A ruthenium(II) complex as turn-on Cu(II) luminescent sensor based on oxidative cyclization mechanism and its application in vivo

Copper ions play a vital role in a variety of fundamental physiological processes not only in human beings and plants, but also for extensive insects and microorganisms. In this paper, a novel water-soluble ruthenium(II) complex as a turn-on copper(II) ions luminescent sensor based on o-(phenylazo)aniline was designed and synthesized. The azo group would undergo a specific oxidative cyclization reaction with copper(II) ions and turn into high luminescent benzotriazole, triggering significant luminescent increasements which were linear to the concentrations of copper(II) ions. The sensor distinguished by its high sensitivity (over 80-fold luminescent switch-on response), good selectivity (the changes of the emission intensity in the presence of other metal ions or amino acids were negligible) and low detection limit (4.42 nM) in water. Moreover, the copper(II) luminescent sensor exhibited good photostability under light irradiation. Furthermore, the applicability of the proposed sensor in biological samples assay was also studied and imaged copper(II) ions in living pea aphids successfully.

A s an essential transition metal ion not only for human beings and plants but also for extensive insects and microorganisms, Cu(II) plays a vital role in a variety of fundamental physiological processes including neurotransmission, energy generation, iron transportation, pigmentation and scavenging of free radicals 1,2 . Moreover, the internal concentrations of Cu 21 in normal organisms are tightly regulated and disruption of the copper homeostasis often cause disease states or pathophysiological conditions 3,4 . For humans, alterations in the cooper homeostasis are connected to some serious neurodegenerative diseases [5][6][7][8] and may cause gastrointestinal disturbance or damages to liver and kidney 9 . While, for insects such as aphids, excess or deficiency in Cu(II) not only hinders their normal growth and development but also affects their plant responses [10][11][12][13] , which is closely related to the damage extent with their host plants. In addition, due to their widespread use in industry and agriculture, cupric ions are also considered to be a significant environmental pollutant 14 . Consequently, developing robust and versatile methods to investigate the biological and environmental roles of copper(II) ions have been attracted extensive attentions.
Among the reported methods for copper ions detection, luminescent probes are extensively employed owing to their distinct advantages in sensitivity and biological imaging 15 . However, due to the intrinsic fluorescence quenching property of Cu 21 stemming from its paramagnetic nature, most hitherto reported Cu 21 sensors have shown a ''turn-off'' response via an electron/energy transfer process [16][17][18] . Although some luminescence ''off-on'' Cu 21 sensors with high selectivity 19,20 , nanomolar sensitivity [19][20][21][22] , good water solubility 20,23 , excellent photostability 24 and long emission wavelength 25,26 have been reported, sensors combining all these features are rare up to now 15 . Furthermore, the probes for Cu 21 detection in biological systems, such as different cancer cells 25,27 , rat hippocampal slices 25,27 , zebrafish 25,28 , human tissues 27,29 , herb leaves 30 have been investigated, however, copper(II) imaging in insects is rarely reported. Definitely, developing new Cu 21 -selective turn-on luminescent probes with excellent performance for diverse biological systems is still of importance and necessity. Ruthenium(II) complexes are one type of potential candidates for environmental and biological Cu 21 probing, due to their good water solubility, high chemical and photostability, intense polarized luminescence, red emission, large Stokes shifts, and long lifetimes 31 . To date, some ruthenium(II) complex based luminescent probes for Cu 21 have been developed 24,[32][33][34][35][36][37][38][39][40][41] . Unfortunately, as far as we know, there is only one example of luminescence enhancement Cu 21 sensor based on a ruthenium(II) complex (Rubp-Ptz) reported by Gopidas's group, which could detect micromolar amounts of Cu 21 in acetonitrile solution 24 . Herein, in this paper, we focus on the development of a turn-on ruthenium(II) complex based luminescent sensor with superior performance for Cu 21 detection and imaging.
Lee et al. reported a fluorescence turn-on chemodosimeter for Cu 21 based on oxidative cyclization of a non-emissive azoaniline into a highly fluorescent benzotriazole product, which can detect mM-level concentrations of Cu 21 in water at room temperature with the green emission 23 . Given the relatively high detection limit of the reported sensor, here, we designed and synthesized a novel ruthenium(II) complex RuMAZO ( Fig. 1) with o-(phenylazo) aniline group as a turn-on luminescent sensor for Cu 21 . The non-emissive RuMAZO in presence of copper(II) ions undergoes oxidative cyclization to form a highly luminescent product RuTAZO (Fig. 1).

Results
The non-emissive RuMAZO in presence of copper(II) ions undergoes oxidative cyclization to form a highly luminescent product RuTAZO (Fig. 1). This cyclization reaction can be triggered by nM-level (4.42 nM) concentrations of Cu 21 in a HEPES (HEPES 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, to exhibit . 80-fold enhancement in a red emission at l max em 5 599 nm. The chloride salt of RuTAZO exhibits l max em value at 599 nm with a quantum yield of 4.7%, using [Ru(bpy) 3 ] 21 (bpy 5 2,2-bipyridine) as a standard (see supplementary information part) 42 . Moreover, this probe has proved to be an appropriate luminescent Cu 21 imaging reagent in live pea aphis. To the best of our knowledge, this is the first report on developing a ruthenium(II) complex-based luminescent sensor for luminescence enhancement detecting Cu 21 in aqueous solution with high selectivity and sensitivity and imaging Cu 21 in insects.
Luminescence enhancement was clearly evident up to 30 min in a HEPES buffer solution (20 mM, pH 7.4, 37uC), then no further significant changes occurred, indicating that the optimal reaction time for Cu 21 detection via oxidative cyclization for this sensor is around 30 min (Fig. S1). In addition, the luminescence properties of RuMAZO were checked under the same conditions. As shown in Fig.  S2, after treatment with different concentrations (0-3 equiv.) of Cu 21 at a physiological temperature 37uC, the ligand absorption of RuMAZO (10 mM) at around 263 nm apparently increased and the metal-to-ligand charge transfer (MLCT) absorption at around 452 nm decreased, whereas a new ligand absorption peak at about 294 nm appeared. Correspondingly, within 30 min of reaction under the same conditions, the emission intensity at 599 nm increased to over 80 folds upon excitation at 465 nm with only 1 equiv. of Cu 21 (Fig. 2a). The Stokes shift of RuTAZO is 134 nm. These results indicate that the o-(phenylazo)aniline group of RuMAZO can be efficiently converted into luminescent benzotriazole. Furthermore, the dose-dependent luminescence enhancement followed a good linear relationship with very low Cu 21 concentrations in the range of 0.1-2.0 mM (Fig. 2b) and the limit of detection (LOD) for Cu 21 with RuMAZO (10 mM) was determined to be 4.42 3 10 29 M (see supplementary information part), lower or comparable to those of most previously reported highly sensitive sensors 15 . Thus, the broad linear range and low detection limit make RuMAZO suitable for environmental or biological copper(II) detection and imaging.
For further biological applications, the cytotoxicity of RuMAZO and Cu 21 to the HeLa cell lines was investigated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after a 24 h treatment (Fig. S10). RuMAZO did not exhibit obvious cytotoxicity towards the HeLa cell lines at the concentrations employed. Confirming RuMAZO can be a suitable luminescence chemosensing probe for Cu 21 detection in vivo.
To investigate the practical applicability of RuMAZO as a Cu 21 sensor in the luminescence imaging of living cells, HeLa cells were incubated with RuMAZO (10 mM) for 2 h at 37uC in a PBS (phosphate buffer solution, pH 5 7.4). After washed with PBS to remove the remaining RuMAZO, no obvious luminescence could be observed from the confocal laser scanning microscopy (Fig. S11a). However, the intracellular luminescence showed a clear red luminescence after incubated with Cu 21 (20 mM) and PDTC (pyrrolidine dithiocarbamate, 100 mM) for 2 h at 37uC (Fig. S11b). PDTC 43 was used to increase the intracellular level of Cu 21 . The results revealed that RuMAZO could be used as an off-on luminescent probe for imaging Cu 21 in living cells.
To examine the applicability of the sensor for visualizing Cu 21 in living organisms, four-day-old pea aphids were selected and divided into three groups. The first two groups were given skin-pop injections at the bottom of the middle legs with Cu 21 (300 nL, 5 mM in a HEPES buffer solution (20 mM, pH 7.4)) or RuMAZO (300 nL, 25 mM in a HEPES buffer solution (20 mM, pH 7.4)) respectively as the control. The third group was given a hypodermic injection of 25 mM RuMAZO and then 50 mM Cu 21 (300 nL, 20 mM HEPES) immediately. All samples were imaged using a Confocal Laser Scanning Microscope with a 488 nm excitation laser after incubation for 6 h. As shown in Fig. 3, pea aphids in the experimental group exhibited distinct luminescence signal over the entire bodies. While no apparent emission was observed in the control groups, illustrating that RuMAZO could detect Cu 21 in vivo without the interference of background signals. Taken together, RuMAZO is proved to be a desired turn on imaging agent for visualizing the distribution of Cu 21 in insects, based on this, a reliable method could be established for investigating the functions of Cu 21 on the plant response of aphids, the work is ongoing now.

Discussion
To investigate the sensing mechanism of RuMAZO to Cu 21 , the reaction product of RuMAZO with Cu 21 in ethanol/H 2 O mixture was isolated and characterized by 1 H NMR, 13 C NMR and HR-MS ( Fig. S19-S21). Furthermore, the isolated product exhibited nearly identical UV-vis and luminescence spectra with those of the testing mixture of RuMAZO and Cu 21 incubated at 37uC for 30 min (Fig.  S3). The result of the EDTA (EDTA 5 ethylene diamine tetraacetic acid) competitive experiment provided further evidence on the nonbinding interaction between RuMAZO and Cu 21 (Fig. S4). All these demonstrated the above mentioned proposed mechanism.
To verify the selectivity of RuMAZO towards Cu 21 , the influence of other metal ions on the sensing of Cu 21 was determined. As shown in Fig. 4 and Fig. S5, the changes of the emission intensity of RuMAZO in the presence of 10.0 equivalents of other metal ions were negligible. Upon the addition of only 1.0 equivalent of Cu 21 to the 1510 mixture of RuMAZO and other metal ions, a significant luminescence enhancement was observed, indicating that the existence of those metal ions in testing samples did not interfere copper(II) detection and imaging. Different copper salts (CuSO 4 , CuCl 2 , Cu(NO 3 ) 2 and Cu(OAc) 2 ) were also tested, not much affection can be observed on the response of RuMAZO to Cu 21 ions with the presence of different counter anions (Fig. S6).
RuMAZO was observed to exhibit good photostability under the irradiation of 500 W iodine-tungsten lamp for 2 h (Fig. S7), this is beneficial for long-time luminescence tracking. In addition, the influence of pH on the luminescence of RuMAZO and RuTAZO    was examined by luminescence titration under different pH value. As shown in Fig. S8, no obvious signal changes were observed over the pH range of 2-13, confirming that the luminescence of RuMAZO and RuTAZO was independent of pH and expected to work well under physiological conditions.
Amino acids were also examined as potential interfering factors for bioimaging applications of the probe. The result demonstrated that the presence of amino acids had no interference with the sensitive detection of Cu 21 by RuMAZO (Fig. S9). All these proved that RuMAZO is appropriate for biological Cu 21 sensing and imaging.
In summary, a fully water-soluble ruthenium(II) complex (RuMAZO) with o-(phenylazo)aniline group as reactive site has been developed as a turn on copper(II) luminescence sensor. Under a physiological environment (20 mM HEPES buffer solution, pH 7.4; 37uC), nonemissive RuMAZO can be efficiently transformed into high luminescent RuTAZO by an oxidative cyclization reaction with Cu 21 within 30 min, which can be triggered by nM-level (4.42 nM) concentration of Cu 21 with excellent selectivity. Moreover, the probe has been employed to image Cu 21 in live pea aphids with a turn-on luminescence signal.

Methods
All solvents and chemical reagents employed for synthesis were analytical grade and purchased from commercial suppliers. The solutions of EDTA and metal ions were prepared from either their chloride or their nitrate salts. Deionized water was used as solvent. HEPES buffered aqueous solution (20 mM, pH 5 7.4) was prepared in double-distilled water. Pea aphids, four-day-old, were obtained from Key Laboratory of Applied Entomology of Northwest A&F University. 1 H NMR and 13 C NMR spectra were recorded on a Bruker 500 AVANCE III spectrometer with chemical shifts reported in ppm at room temperature. Mass spectra were obtained with Thermo Fisher LCQ Fleet mass spectrometer (USA) and a LC/Q-Tof MS spectrometry (USA). The pH of the testing systems was determined by a PHS-3C pH Meter (China). Absorption spectra were collected by using a Shimadzu 1750 UV-visible spectrometer (Japan). Emission spectra were measured with a Shimadzu RF-5301 fluorescence spectrometer (Japan). Microinjection experiments were carried out by using a Drummond Nanoject II TM Auto-Nanoliter Injector (USA). Images of pea aphids were performed on an Olympus FV1000 confocal microscope (Japan).
Compound 1 was prepared according to the literature 44 . The MAZO ligand was prepared through the coupling reaction of compound 1 with phenyl diazonium salt 45 . The ruthenium(II) complex was obtained in a satisfactory yield (89%) through direct reaction of MAZO with the appropriate molar ratios of cis-[Ru(phen) 2 Cl 2 ] in ethanol 46 .
MAZO. Aniline (186 mg, 2.0 mmol) was dissolved in 2 mL concentrated hydrochloric acid, then 8 mL cold solution of NaNO 2 (138 mg, 2.0 mmol) was added. The mixture was stirring under 0uC for 1 h. Then it was added into 36 mL 5amino-1,10-phenanthroline (400 mg, 2.05 mmol) acetate buffer (3.0 g sodium acetate and 6 mL acetate) in dropwise in 30 minutes. After the addition was complete, the mixture was stirred for 24 h. Then mixture was filtered and the filtrate was suspended in 50 mL 3% ammonia. Stirred overnight and then filtered, washed with pure water, recrystallized in absolute ethanol. The yield was 0.548 g, 89.4%. 1