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1 Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 214 Supporting information Materials and General Instruments Doubly purified water used in all experiments is from Milli-Q systems. Other solvents and reagents were of analytical grade and used without further purification. Stock of ct-da, chymotrysin, lysozyme, human serum albumin (HSA), protease and bovine serum albumin (BSA) were obtained from Sigma Chemical Co. (USA). RA (S. cerevisiae RA) was purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. Hoechst 33258, SYTO RAselect were purchased from Molecular Probes. Dase and Rase were purchased from Sigma. 1 H MR and 13 C MR spectra were recorded on a VARIA IOVA-4 spectrometer with chemical shifts reported as ppm. Mass spectrometric data were obtained on a Q-Tof MS spectrometer (Micromass, Manchester, England). Absorption spectra were measured on a Perkin-Elmer Lambda35 UV-Vis spectrophotometer. Luminescence measurements were performed on a VARIA CARY Eclipse luorescence Spectrophotometer. General Methods All reactions were carried out under a nitrogen atmosphere. Silica gel (2-3 mesh) was used for flash column chromatography. Phosphate buffered saline (PBS) (ph = 7.5, 2 mm) was prepared using doubly purified water. Stock of ct-da, chymotrysin, lysozyme, human serum albumin (HSA), protease and bovine serum albumin (BSA) are prepared by dissolving them in doubly purified water. The concentration of ct-da is determined spectrophotometrically using the molar absorption coefficients of ε26 nm = 66M 1.cm 1. 1 mm stock solutions of, RuRu and were prepared in DMSO for the absorption and emission spectral measurements. In the absorption and emission titration studies, the solutions of the compounds (2 μm) are titrated with aliquots of RA solutions to reach the different molar ratio to the dyes. Concentration of RA in working solutions was 1 mm. Photostability of in solution and SYTO RA-select were dissolved in DMSO at a concentration of 1. μm, respectively. The solutions were irradiated under a 5W iodine-tungsten lamp for 25 min at a distance of 25 mm away. An aqueous solution of sodium nitrite (5. g/l) as heat filter was placed between the samples and the lamp as a light filter (to cut off the light shorter than 4 nm). The photostabilities were expressed in the terms of remaining absorption (%) calculated from the changes of emission at the emission maximum before and after irradiation by iodine-tungsten lamp. The emission was determined on VARIA CARY Eclipse luorescence Spectrophotometer. Interaction of with Biomolecules The interaction of with RA, DA Chymotrysin, Lysozyme, BSA, HSA and Protease was carried out in a phosphate buffered saline (PBS) (ph=7.5, 2 mm) using luminescence detection. The emission was determined on VARIA CARY Eclipse luorescence Spectrophotometer. Cell culture and staining MC-7 and Hella and LO2 cells were cultured in DEME (Invitrogen) supplemented with 1% CS (Invitrogen). One day before imaging, cells were seeded in 24-well flat-bottomed plates. The next day, the live cells were incubated with 2 µm, RuRu and for 2 h at 37 C under 5% CO 2 and then washed with phosphatebuffered saline (PBS) three times. luorescence imaging was then carried out with a TCS-SP2 confocal laser scanning microscope (Leica, Germany), using a 1 objective lens. Before Dase and Rase digest and colocalization experiments, cultured cells grown on special confocal microscope dish were fixed by precooled methanol (-2 C) for 15 min, washed with PBS for 5 min twice. or Dase and Rase digest test, three sets of pretreated MC-7 cells were stained with 2 μm for 1 min. A total of 1 μl clean PBS (as control experiment), 3 μg/ml Dase (Sigma), or 25 μg/ml Dase-ree Rase (GE) was added into the three adjacent wells and incubated at 37 ºC in 5% CO 2 for 1 h. Cells were rinsed by clean PBS twice more before imaging. or each dye test, the fluorescent imaging pictures were obtained with an equal parameter for control. or cells colocalization experiment: fixed MC-7 cells were stained with 2 μm for 1 min. After rinsing with PBS twice, the same sample was stained with 2 μm Hoechst and SYTO RA-select for 1 min and then imaged. Synthetic procedures Synthesis of metal complexes [Ru(bpy) 3 -(CH 2 ) 1 -Ru(bpy) 3 ] 4+ and [( 2 ppy) 2 -(CH 2 ) 1 -( 2 ppy) 2 ] 2+. The dimetallic complexes [Ru(bpy) 3 - (CH 2 ) 1 -Ru(bpy) 3 ] 4+ and [( 2 ppy) 2 -(CH 2 ) 1 -( 2 ppy) 2 ] 2+ were synthesized using a published method S1, 27, 28 with suitable modification. [Ru(bpy) 3 -(CH 2 ) 1 -( 2 ppy) 2 ] 3+. The syntheses of [Ru(bpy) 3 -(CH 2 ) 1 -( 2 ppy) 2 ] 3+ were accomplished as described in Scheme S1. Under nitrogen atmosphere, [Ru(bpy) 3 ]Cl 2 (231 mg,.44 mmol) and bpy-c 1 -bpy (2 mg,.44 mmol) were refluxed in a mixture ethanol-h 2 O (8:1, 45 ml) for 24 h. After cooled and solvent removal, purification by preparative tin layer chromatography using as eluent the mixture CH 3 COCH 3 /H 2 O/saturated KO 3 solution (15/1/1) gave Ru-C 1 -bpy (386 mg, 7%). Then a mixture of ( 2 ppy) 2 (m-cl) 2 ( 2 ppy) 2 (3 mg,.24 mmol) and Ru-C 1 -bpy (554 mg,.48 mmol) were heated to reflux in a dichloromethane-ethanol mixture (3:1, 25 ml) for 4 h. After the solution was cooled to room temperature and filtered, a solution of H 4 P 6 (3.2 g, 19.6

2 mmol) in water (ca. 2 ml) was added slowly to the filtrate. The resulting precipitate was collected by filtration. The solid was washed with CHCl 3 and diethyl ether. The solid was dried at 8 C for 24 h to afford the desired compound as a red solid (826 mg, 92%). 1 H-MR (4 MHz, δ ppm, Acetone-d 6 ): (m, 12H), (m, 4H), (m, 4H), 5.79 (dd, J = 8.5, 2.3 Hz, 2H), (m, 2H), 7.24 (m, 2H), 7.45 (dd, J = 5.9, 1.6 Hz, 1H), (m, 6H), (m, 1H), (m, 3H), (m, 8H), (m, 6H), (m, 3H), (m, 8H). 13 C MR (1 MHz, Acetone-d 6 ): δ 29.99, 3.6, 34.92, 35.11, 98.66, , , , , , 124.4, , 125.2, , , , , , , , , , , 14.9, , , 15.31, 15.95, 151.5, , , , , , , , , , , , , HRMS (ESI, m/z): 1/2 (M-2P 6- ) + calculated for C 72 H P , found Cl 3 nh 2 O C Cl Cl C ppy ( 2 ppy) 2 ( -Cl) 2 ( 2 ppy) 2 1 bpy-c 1 -bpy Ru(bpy) 2 Cl 2 2H 2 O Ru 1 Ru-C 1 -bpy ( 2 ppy) 2 ( -Cl) 2 ( 2 ppy) 2 Ru 1 Scheme S1. The synthesis of. Ru 1 Ru 4P 6 RuRu 1 C C 2P 6 ig. S1 The chemical structures of RuRu and.

3 ig. S2 Emission intensity decay for (1. μm) in acetonitrile at room temperature. ig. S3 Emission intensity decay for SYTO RA-select (1. μm) in acetonitrile at room temperature. / / % SYTO RA-Select Time / min 2 25 ig. S4 Photostabilities of and SYTO RA-select in solution.

4 RuRu ig. S5 The luminescent spectra of RuRu (2. µm) in phosphate buffered saline (PBS) (ph=7.5, 2 mm) at 298 K with the addition of RA ig. S6 The luminescent spectra of (2. µm) in phosphate buffered saline (PBS) (ph=7.5, 2 mm) at 298 K with the addition of RA.

5 ig. S7 The luminescent spectra of (2. µm) in phosphate buffered saline (PBS) (ph=7.5, 2 mm) at 298 K with the addition of RA and the ratiometric luminescence response between (2. µm) to RA, Ratio means [RA]/[ ], I /I Ru stands for the ratio of the luminescence intensity of iridium moiety (523 nm) and ruthenium moiety (615 nm).. ( I min -I) /( I min -I max ) Y= *X R= Log[ RA] ig. S8 A linear calibration curve between the I /I Ru ratio and RA amount, I refers to I /I Ru.

6 RA +DA ig. S9 Emission spectra of (2. µm) in the presence and absence of 8-fold excess RA and DA. I / I Ru RA DA Chymotrysin Lysozyme BSA HSA Protease ig. S1 Emission spectra of (2. µm) in phosphate buffered saline (PBS) (ph=7.5, 2 mm) at 298 K in the presence and absence of 8-fold excess RA, DA, chymotrysin, lysozyme, BSA, HSA and protease. I /I Ru stands for the ratio of the luminescence intensity of iridium moiety (523 nm) and ruthenium moiety (615 nm). a) tra b) 7 6 +total RA ig. S11 a) Emission spectra of (.33 μm) in phosphate-buffered saline (PBS; ph 7.5, 2 mm) in the presence or absence of 8-fold excess tra. b) Emission spectra of (2. μm) in phosphate-buffered saline (PBS; ph 7.5, 2 mm) in the presence or absence of 8-fold excess total RA.

7 a) SYTO RA-Select SYTO RA-Select+tRA b) 3 SYTO RA-Select 25 SYTO RA-Select+yeast RA ig. S12 a) Emission spectra of SYTO RA-Select (5. μm) in phosphate-buffered saline (PBS; ph 7.5, 2 mm) in the presence or absence of 2-fold excess tra. b) Emission spectra of SYTO RA-Select (5. μm) in phosphate-buffered saline (PBS; ph 7.5, 2 mm) in the presence or absence of 2-fold excess yeast RA poly(da-dt) +poly(dg-dc) +poly(da-du) +poly(du) ig. S13 Emission spectral of complex (2 μm) in PBS (ph=7.5, 2 mm) at 298 K in the presence of 2-fold excess poly(da-dt), poly(dg-dc), poly(da-du) and poly(du) eq KI +4eq KI +6eq KI +8eq KI +1eq KI +12eq KI +14eq KI RA +RA+2eq KI +RA+4eq KI +RA+6eq KI +RA+8eq KI +RA+1eq KI ig. S14 Emission spectra of complex (.33 μm) alone and with the presence of 6 eq RA in phosphate buffered saline (PBS) (ph=7.5, 2 mm) with the addition of KI.

8 eq acl +4eq acl +6eq acl +8eq acl +1eq acl RA +RA+2eq acl +RA+4eq acl +RA+6eq acl ig. S15 Emission spectra of complex (.33 μm) alone and with the presence of 6 eq RA in phosphate buffered saline (PBS) (ph=7.5, 2 mm) with the addition of acl. ig. S16 Luminescence microscope images of live a) Hella cells b) and LO2 cells incubated with 2 μm for 2h at 37 C. Scale bar: 2 μm. ig. S17 a) Incubation of live MC-7 cells with RuRu (2 μm, 2 h) shows no staining of live cells. The red channel was stained with RuRu with excitation at 458 nm and a scan range of nm. b) Live MC-7 breast cancer cells incubated with (2 μm, 2 h). The green channel was stained with with excitation at 45 nm and a scan range of nm. rom left to right: Optical, luminescence, and overlay images. Scale bar : 2 μm.

9 ig. S18 Treatment of fixed MC-7 cells with Dase and Rase. was used at a concentration of 2 µm. rom left to right: green channel (5 56 nm), red channel (59 64 nm), and overlay images. Scale bar: 2 μm. ig. S19 Colocalization of (2 μm) and a) SYTO RA-Select dye (5 μm) and b) Hochest33258 (5 μm), images by fixed MC-7 cell loading. a) Co-staining of with SYTO RA-Select dye. Left, RA-Select dye (green channel: nm; excitation at 45 nm); middle, luminescence (red channel: nm; excitation at 458 nm); right, merged image. b) Colocalization of and Hochest Left, Hochest33258 (blue channel: nm; excitation at 45 nm); middle, luminescence (red channel: nm; excitation at 458 nm); right, merged image. Scale bar: 2 μm. [S1] M. Warner, L. Wallace,. R. Keene, J. G. Collins, Dalton Trans. 213, 42,