Turn-on Luminescent Probe for Hydrogen Peroxide Sensing and Imaging in Living Cells based on an Iridium(III) Complex–Silver Nanoparticle Platform

A sensitive turn-on luminescent sensor for H2O2 based on the silver nanoparticle (AgNP)-mediated quenching of an luminescent Ir(III) complex (Ir-1) has been designed. In the absence of H2O2, the luminescence intensity of Ir-1 can be quenched by AgNPs via non-radiative energy transfer. However, H2O2 can oxidize AgNPs to soluble Ag+ cations, which restores the luminescence of Ir-1. The sensing platform displayed a sensitive response to H2O2 in the range of 0−17 μM, with a detection limit of 0.3 μM. Importantly, the probe was successfully applied to monitor intracellular H2O2 in living cells, and it also showed high selectivity for H2O2 over other interfering substances.

The sensing mechanism of the Ir-1-AgNP probe for H 2 O 2 is illustrated in Fig. 1. In the initial system, the luminescence of Ir-1 was significantly quenched by AgNPs. However, this AgNPs-induced quenching effect can be reversed by H 2 O 2 due to oxidation of AgNPs to Ag + . To our knowledge, the Ir-1-AgNP is the first application of the combination of Ir(III) complexes and AgNPs for H 2 O 2 sensing in both aqueous solutions and living cells.

Results and Discussion
Sensing Mechanism. Ir-1, carrying tfppy as its C^N ligand and pyphen as its N^N ligand (Fig. 2a), was characterized by 1 H-NMR, 13 C-NMR and HRMS (Figs S1-S3 and Table S1). Ir-1 emits strong luminescence at 545 nm under the excitation of 295 nm in aqueous buffer solution. As expected, the luminescence of Ir-1 decreased gradually with increasing amounts of AgNPs in solution (Fig. 2b). This is because the positively charged Ir-1 could be adsorbed on the surface of the citrate-stabilized AgNPs through electrostatic interactions, which efficiently quenched the luminescence of Ir-1. However, the luminescence could be recovered in the presence of H 2 O 2 attributed to oxidation of AgNPs into soluble Ag + by H 2 O 2 . In order to study the kinetic behavior between the Ir-1-AgNP system and H 2 O 2 , the luminescence change was monitored as a function of time. As shown in    . S4, the luminescence intensity of the Ir-1-AgNP system increased with time and reached the plateau after 10 min, indicating that the reaction between AgNPs and H 2 O 2 at ambient temperature is rapid. In the absence of AgNPs, H 2 O 2 showed no apparent effect on the luminescence of Ir-1 (Fig. S5). Therefore, the increase in the luminescence of the system should arise primarily from the decomposition of AgNPs by H 2 O 2 , which restores the emission of Ir-1.
The mechanism involved in the luminescence quenching and recovery process was also demonstrated by transmission electron microscopy (TEM) imaging. In the absence of the Ir-1, the AgNPs were well-dispersed (Fig. 3a). However, after the addition of Ir-1, slight aggregation of AgNPs was observed, suggesting that Ir-1 and AgNPs interacted on the surface of AgNPs (Fig. S6b). The identity of the Ir-1-AgNP complex was further confirmed by energy dispersive X-ray spectroscopy (EDX), which showed strong elemental signals for both Ir and Ag (Fig. S6c). Strikingly, after treatment of AgNPs with H 2 O 2 , no AgNPs could be observed in the TEM images (Fig. 3b). This suggests that the AgNPs were decomposed and transformed to Ag + , which is consistent with previously reported [36][37][38] . The UV-vis absorbance spectra of AgNPs in the absence and presence of H 2 O 2 are shown in Fig. S7. AgNPs alone showed a strong characteristic surface plasmon resonance peak at around 390 nm 39 . However, the absorption band of AgNPs gradually decreased upon increasing concentration of H 2 O 2 . These phenomena were ascribed to the oxidation of AgNPs to Ag + by H 2 O 2 , leading the decomposition of the AgNPs.

Sensitivity.
To explore the applicability of the proposed luminescence sensor for H 2 O 2 detection, we studied the luminescence response of the Ir-1-AgNP system toward varying concentrations of H 2 O 2 . The luminescence intensity of the system was gradually restored with increasing concentration of H 2 O 2 (Fig. 4a). Meanwhile, a good linear relationship over the range from 0 to 17 μmol L −1 with a correlation coefficient of 0.998 was obtained (Fig. S8). The limit of detection (LOD) was calculated to 0.3 μM according to the signal-to-noise method

Selectivity.
To assess the selectivity of Ir-1-AgNPs system for H 2 O 2 , the influences of metal ions and amino acids were studied. As shown in Fig. 4b, nearly no luminescence changes could be observed with the other substances (Fig. 4b), which demonstrates that the Ir-1-AgNP system is highly selective for H 2 O 2 over other non-target substances.
Cell imaging. Given the promising capability of Ir-1 for sensing H 2 O 2 in aqueous solution, we then investigated the ability of Ir-1 for monitoring H 2 O 2 in living human cells. Ir-1 showed cytotoxicity against HeLa (human cervical cancer) cells with an IC 50 value of 5.12 μM (Fig. S9).
In the cell imaging study, the luminescence intensity of HeLa cells was enhanced with increasing concentration of Ir-1 (Fig. 5a), showing that Ir-1 could effectively penetrate into cells. A concentration of 0.3 μM of Ir-1 was chosen for subsequent cell experiments as this concentration was over 10-fold lower than the IC 50 value for cytotoxicity, while it still gave a good luminescence signal.
Next, HeLa cells were pretreated with Ir-1 (0.3 μM) for 1 h before incubation with different concentration of AgNPs. The luminescence intensity of HeLa cells was remarkably reduced with increasing concentration of AgNPs (Fig. S10), which was attributed to AgNPs-mediated quenching of an luminescent Ir-1 as described previously. However, when H 2 O 2 was added into the growth medium for another 1 h, the luminescence of HeLa cells was recovered in a dose-dependent manner (Fig. 5b). Collectively, these results suggest that Ir-1-AgNP can be developed for the monitoring of H 2 O 2 levels in living cells.

Conclusion
Consequently, we have proposed a turn-on luminescence assay for H 2 O 2 detection employing the Ir-1-AgNP system. In this nano-composite system, Ir-1 functioned as a luminescence reporter, while AgNPs were employed both as a luminescence quencher and as a recognition unit for H 2 O 2 . Based on the luminescence recovery of the Ir-1-AgNP system triggered by H 2 O 2 , this nanoprobe was successfully applied to detect H 2 O 2 at the intracellular level in living cells. In addition, the Ir-1-AgNP probe possesses some superior properties, including label-free, good sensitivity and selectivity, low cost, easy manipulation, low cytotoxicity, and turn-on luminescent response. To our knowledge, the probe is the first combination of Ir(III) and AgNPs applied for the detection of H 2 O 2 in living cells reported in the literature.

Materials and Methods
Chemicals and materials. Iridium chloride hydrate (IrCl 3 ·xH 2 O) was purchased from Precious Metals Online (Australia). Other reagents were purchased from Sigma Aldrich (St. Louis, MO) and used as received. All of the reagents were of analytical grade and were used as received without further purification. All solutions were prepared in Milli-Q water under ambient conditions. HeLa cell lines were obtained from ATCC (Manassas, VA, USA). Dulbecco's Modified Eagle's medium, fetal bovine serum, penicillin and streptomycin were obtained from Sigma-Aldrich Co. LLC (St. Louis, MO, USA).

Synthesis of AgNPs.
AgNPs were fabricated according to reported methods with slight modifications 28,45 .
In a typical procedure, 0.08 mL AgNO 3 (0.1 M) and 0.1 mL trisodium citrate (0.1 M) were mixed into 100 mL pure water and stirred under the condition of ice bath. Then, freshly prepared NaBH 4 solution was added dropwise into the mixture until it turned yellow. The resulting yellow solution was stirred for another 30 min to form AgNPs quantitatively, which was stored at 4 °C for subsequent use. The diameter of AgNPs prepared was measured to be 8-9 nm by transmission electron microscopy (TEM).
Synthesis of Ir-1. Ir-1 was synthesized based on a reported literature method 46-49 . [Ir 2 (tfppy) 4 Cl 2 ] (0.2 mmol) and pyppy (0.42 mmol) in a mixed solvent of DCM:methanol (1:1.2 (v/v), 36 mL) was refluxed overnight. The reaction mixture was allowed to cool to ambient temperature, and unreacted cyclometallated dimer was removed by filtration. Excess ammonium hexafluorophosphate was then added into the filtrate, and the resulting mixture was stirred for another 30 min. Afterwards, the solution was evaporated under reduced pressure until precipitation was initiated. The precipitate was filtered, and washed by several portions of water and diethyl ether. The crude product was then recrystallized by the acetonitrile/diethyl ether vapor diffusion to obtain the desired compound, which was characterized by 1 H-NMR, 13 C-NMR, high resolution mass spectrometry (HRMS) and elemental analysis. Cell imaging. HeLa cells were pretreated with Ir-1 (0.3 μM) for 1 h at 37 °C, then AgNPs of different concentrations (0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM and 5 μM) was added before further incubation for 1 h. After washing with PBS three times, the luminescence intensity of HeLa cells was imaged by a Leica SP8 laser scanning confocal microscope upon excitation at 405 nm.
For H 2 O 2 detection, the experiment was performed as above except that after incubation in the presence of AgNPs (2.8 μM), cells were further treated with H 2 O 2 ranging from 0 to 20 μM for 1 h. After washing with PBS three times, the luminescence intensity of HeLa cells was then imaged as above.
Statistics analysis. One-way analysis of variance (ANOVA) followed by the Dunnett's method for multiple comparisons by using GraphPad Prism 6.0 was used to analyse the data.