Supplemental Information. Two-Photon Luminescent Bone Imaging. Using Europium Nanoagents

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1 Chem, Volume 1 Supplemental Information Two-Photon Luminescent one Imaging Using Europium Nanoagents Esther M. Surender, Steve Comby, renton L. Cavanagh, Orlaith rennan, T. Clive Lee, and Thorfinnur Gunnlaugsson

2 SUPPLEMENTL EXPERIMENTL PROCEDURES 1. General Experimental Procedures Procedure 1: Synthesis of Ln(III) complexes using Ln(CF3SO3)3 ll Ln(III) complexes found within were prepared by refluxing, under microwave irradiation, the relevant ligand with 1. eq. of Ln(CF3SO3)3 for 3 hr in freshly distilled CH3OH. The solvent was then reduced to ca. 1 ml and the relevant complexes were isolated by precipitation from swirling dry diethyl ether (2 ml). Owing to the paramagnetic nature of the Ln(III) ion, 1 H NMR spectra of the complexes consisted of broad resonances and, therefore, were not fully characterized in terms of integration. These paramagnetic properties also prevented 13 C NMR spectra from being recorded. Procedure 2: ase hydrolysis of the diethyliminodiacetate functional groups 1,2 lkaline hydrolysis was carried out by refluxing, under microwave irradiation, the relevant Ln(III) complex with 6. eq. of NaOH for 24 hr in a mixed CH3OH/H2O (1:9 v/v) solution. fter reaction completion, the solvent was reduced to ca. 5 ml and acidified to ph 3 using 2M HCl. The supernatant was then decanted and the protonated product was dissolved in either CH3CN or IP. The precipitated salts were subsequently removed by centrifugation and the hydrolyzed Ln(III) complex, in its acid form, was afforded upon removal of the solvent by reduced pressure. Lastly, the corresponding sodium carboxylate form of the complex was then given by adding 6. eq of aqueous NaOH. Procedure 3: Gold nanoparticle synthesis 3,4 u(iii) chloride trihydrate (.1 g,.25 mmol) was dissolved in Millipore H2O (1 ml) and tetraoctylammonium bromide (.36 g,.26 mmol) in toluene (25 ml). The two solutions were then stirred vigorously at room temperature for 1 min. NaH4 (.12 g, 3.17 mmol) dissolved in Millipore H2O (1 ml) was then added slowly using a pressure equalized dropping funnel, and the resulting solution was stirred at room temperature for a further 2 hr. Following successful transfer of the u() into the toluene layer, the organic and aqueous layers were separated, and the toluene layer was washed with H2O (2 2 ml),.1 M HCl (2 2 ml), and.1 NaOH (2 2 ml). Formation of the TO-stabilized unps was confirmed by UV-vis absorption spectroscopy by the SPR band observed at 525 nm in toluene. 3-5 Procedure 4: Surface functionalization of unps with Ln(III) complexes 3,4 unp functionalization was carried out by vigorously stirring a solution of the TO-stabilized unps (5 ml) and the relevant aqueous Ln(III) solution (2.76 mm, 5 ml) in the presence of NaH4 (15.74 mm, 5 μl) at room temperature for 16 hr. Following successful transfer of the unps into the aqueous layer, the two layers were separated, and the aqueous layer was filtered through a PDVF.45 μm microsyringe to give a clear purple solution. ny unbound Ln(III) complex was then removed by either sephadex G-15 or LH-2 column chromatography, using Millipore H2O and HPLC grade CH3OH, respectively, as the eluent. UV-vis absorption spectroscopy verified surface functionalization, with a blue-shift in the SPR band being observed relative to the TO-stabilized unps. 3-5

3 2. Experimental Details Scheme S1. Full synthetic schematic of 1 (free ligand) and the corresponding complex 1.Eu, followed by alkaline hydrolysis to give complex 1.Eu.Na. 11-romoundecane-1-thiol (2) 6 cetyl chloride (6.34 g, 8.77 mmol) was added slowly to a stirred C CH3OH (4 ml) solution. S-(11-bromoundecyl)thioacetate (1. g,.32 mmol) was then added slowly along with CH3OH (1 ml) using a pressure equalised dropping funnel, and the resulting solution was stirred at room temperature for 16 hr. The solvent was then reduced to ca. 25 ml, washed with H2O (1 3 ml), and extracted with CH2Cl2 (2 3 ml). The combined organic layers were dried over MgSO4, filtered, and the solvent removed under reduced pressure to yield 2 as a white solid (.8 g, 3.1 mmol, 93% yield). HRMS (m/z, MLDI): Calculated for C11H23Sr m/z = [2M 2H] +. Found m/z = ; 1 H NMR (4 MHz, CDCl3) δh: 3.4 (2H, t, J = 6.89, CH2r), 2.52 (2H, q, J = 7.2, CH2SH), 1.85 (2H, qu, J = 7.2, CH2CH2r), 1.6 (2H, qu, J = 7.5, CH2CH2SH), (14H, m, 7 CH2); 13 C NMR (4 MHz, CDCl3) δc: (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2); IR υmax (cm 1 ): 2925, 2859, 146, 1261, 199, 129, 95, 84, 731.

4 1,2-is-(11-bromoundecyl)disulfane (3) 6 Compound 2 (.6 g, 2.27 mmol) was dissolved along with iodine (.17 g,.68 mmol) in CH2Cl2 (5 ml), and the resulting red solution was refluxed for 16 hr. fter reaction completion, the organic solution was washed with H2O (3 3 ml), dried over MgSO4, filtered, and the solvent removed under reduced pressure. The product, 3, was obtained as a clear viscous oil (.57 g, 1.8 mmol, 94% yield). HRMS (m/z, MLDI): Calculated for C22H44S2r2 m/z = [M] +. Found m/z = ; 1 H NMR (4 MHz, CDCl3) δh: 3.41 (4H, t, J = 6.91, 2 CH2r), 2.68 (4H, t, J = 7.41, 2 CH2S), 1.85 (4H, qu, J = 7.29, 2 CH2CH2r), 1.6 (4H, qu, J = 7.5, 2 CH2CH2S), (28H, m, 14 CH2); 13 C NMR (4 MHz, CDCl3) δc: (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2); IR υmax (cm 1 ): 2916, 285, 1463, 128, 1225, 119, 958, 885, 717, 64. 1,2-is[11-(1,4,7,1-tetraazacyclododecan-1-yl)undecyl]disulfane (4) 7 Cyclen (1.19 g, 6.91 mmol) was dissolved along with NEt3 (.21 g, 2.8 mmol) in freshly distilled CHCl3 (5 ml). fter stirring the solution for 5 min, 3 (.46 g,.82 mmol) was added and the resulting solution was refluxed for 16 hr under an inert atmosphere. fter cooling to room temperature, the organic solution was washed with 1 M NaOH (3 2 ml) to remove the excess cyclen and with H2O (3 1 ml). The organic layer was dried over MgSO4, filtered, and the solvent removed under reduced pressure to yield the product 4 (.42 g,.59 mmol, 72% yield) as a yellow oil. HRMS (m/z, ESI + ): Calculated for C38H83N8S2 m/z = [M+H] +. Found m/z = ; 1 H NMR (4 MHz, CDCl3) δh: (32H, m, cyclen-ch2), 2.38 (4H, t, J = 7.26, NCH2), 1.63 (4H, q, J = 7.4, CH2CH2S), (36H, m, 18 CH2); 13 C NMR (4 MHz, CDCl3) δc: (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), (CH2), 28.7 (CH2), (CH2), (CH2), (CH2); IR υmax (cm 1 ): 292, 285, 1669, 1456, 1351, 1272, 1112, 15, 935, 727, 61. Diethyl 2,2-(2-chloroacetylamino)diacetate (5) 7 Diethyl iminodiacetate (3. g, mmol) and NEt3 (2.55 g, 25.2 mmol) were dissolved in CH2Cl2 (5 ml). The clear solution was cooled to C and chloroacetyl chloride (3.54 g, mmol) was added slowly along with CH2Cl2 (3 ml) using a pressure equalised dropping funnel. The resulting solution was then stirred at room temperature for 24 hr. Following reaction completion, the pale yellow solution was washed with H2O (2 3 ml),.1 M HCl (2 3 ml), and brine (2 3 ml). The organic layer was dried over MgSO4, filtered, and the solvent removed under reduced pressure to afford 5 as an orange oil (3.87 g, mmol, 92% yield). HRMS (m/z, ESI + ): Calculated for C1H16NO5NaCl m/z = [M+Na] +. Found m/z = ; 1 H NMR (4 MHz, CDCl3) δh: 4.22 (8H, m, CO2CH2CH3), 4.12 (2H, s, COCH2Cl), 1.3 (3H, t, J = 7.2, CH2CH3), 1.27 (3H, t, J = 7.2, CH2CH3); 13 C NMR (4 MHz, CDCl3) δc: (qt), (qt), (qt), (CH2), (CH2), 5.48 (CH2), (CH2), 4.6 (CH2), 14.1 (CH3), 14.8 (CH3); IR υmax (cm 1 ): 2986, 1738, 1661, 1463, 147, 1375, 1299, 126, 1188, 163, 966, 931, 866, 796, 736.

5 1,4,7-Tris-(N,N-bis(ethoxycarbonylmethyl)-1-[dodecane-1-thiol]-1,4,7,1 tetraazacyclododecane (1) Ligand 4 (.2 g,.28 mmol) was dissolved in freshly distilled CH3CN (2 ml) in the presence of Cs2CO3 (.6 g, 1.84 mmol) and KI (.31 g, 1.87 mmol). Compound 5 (.47 g, 1.77 mmol) was then added and the resulting solution was exposed to microwave irradiation for 24 hr at 85 C with 3 sec pre-stirring. fter cooling, removal of the inorganic salts was achieved by centrifugation and the solvent was then reduced to ca. 1 ml. Following NaH4 (.21 g,.56 mmol) was added, and the solution was stirred at room temperature for 5 hr under an inert atmosphere. fter completion of the reaction the solvent was removed under reduced pressure. The resulting orange oil was purified by flash silica column chromatography using a gradient elution of 1 to 8:2 CH2Cl2:CH3OH. The desired product, 1, was obtained as a viscous orange oil (.21 g,.2 mmol, 36% yield). HRMS (m/z, ESI + ): Calculated for C98H174N14O3S2 m/z = [2M+2H] 2+. Found m/z = ; 1 H NMR (6 MHz, CDCl3) δh: (24H, m, (CH2CO2CH2CH3)2), (26H, br m, cyclen-ch2, NCH2CO, NCH2(CH2)9CH2SH), 1.61 (2H, m, (CH2)9CH2CH2SH), 1.23 (34H, m, (CH2)8(CH2)2SH, (CO2CH2CH3)2); 13 C NMR (6 MHz, CDCl3) δc: (qt), (qt), (qt), (qt), (qt), (qt), (qt), (qt), (CH2), (CH2), (CH2), 6.38 (CH2), 6.8 (CH2), (CH2), (CH2), 52.4 (CH2), 5.49 (CH2), 5.9 (CH2), (CH2), 48.9 (CH2), 39.9 (CH2), (CH2), 29.2 (CH2), (CH2), (CH2), (CH2), 21.3 (CH2), (CH3); Calculated for C49H87N7O15S.1CHCl3.1CH2Cl2.1H2O: C, 48.28; H, 7.31; N, Found C, 48.29; H, 7.23; N, 7.8; IR υmax (cm 1 ): 2924, 2855, 1736, 1671, 1597, 1558, 1457, 146, 1369, 133, 119, 199, 123, 876, 747, 66. Complex 1.Eu Complex Eu.1 was synthesised according to Procedure 1 using ligand 1 (.15 g,.14 mmol) and Eu(CF3SO3)3 (.94 g,.16 mmol). viscous yellow oil was obtained (.19 g,.12 mmol, 79% yield). HRMS (m/z, MLDI): Calculated for C5H87N7O18F3S2Eu m/z = [M+CF3SO3] +. Found m/z = ; 1 H NMR (4 MHz, CD3OD) δh: 33.86, 26.6, 23.81, 21.76, 2.4, 19.62, 19.3, 17.76, 17.18, 12.2, 4.93, 4.28, 3.78, 3.38, 3.35, 2.73, 1.71, 1.37,.39, 5.63, 6.64, 7.61, 8.98, 9.86, 1.41, 11.32, 12.39, 14.44, 15.32, 16.49, 19.13, 19.81; IR υmax (cm 1 ): 2929, 286, 1742, 1612, 1535, 1457, 1375, 1224, 1163, 181, 123, 84, 725, 628. Complex 1.Eu.Na Complex Eu.1.Na was synthesised according to Procedure 2 using Eu.1 (.19 g,.11 mmol) and NaOH (.28 g,.7 mmol). The desired product was obtained as a pale yellow solid (.18 g,.12 mmol, 69% yield). m.p. decomposed above 26 C; HRMS (m/z, MLDI): Calculated for C37H61N7O15Eu m/z = [M 6Na+4H]+. Found m/z = ; 1H NMR (4 MHz, D2O) δh: 7.88, 7.45, 7.11, 6.89, 4.92, 3.59, 3.28, 2.94, 2.65, 2.51, 1.99, 1.27,.64,.6,.77; IR υmax (cm 1 ): 2922, 2859, 166, 154, 157, 141, 1236, 178, 118, 978, 842, 79, 631.

6 3. Characterization Figure S1. 1 H NMR (6 MHz) of ligand 1 in CDCl3. Figure S2. 13 C NMR (6 MHz) of ligand 1 in CDCl3.

7 % % Figure S3. 1 H NMR (4 MHz) of complex 1.Eu in CD3OD Calculated Experimental m/z Figure S4. The HRMS isotopic distribution pattern for complex 1.Eu. () Calculated pattern. () Observed pattern.

8 % % Figure S5. 1 H NMR (4 MHz) of complex 1.Eu.Na in D2O Calculated Experimental m/z Figure S6. The HRMS isotopic distribution pattern for complex 1.Eu.Na. () Calculated pattern. () Observed pattern.

9 Intensity (a.u.) Intensity (a.u.) Time (ms) Time (ms) Figure S7. Luminescence lifetime decay of 1.Eu.Na fitted with a mono-exponential function. () Measured in H2O. () Measured in D2O. C Figure S8. Reduction of u(iii) to u() using a modified rust-schiffrin two-phase transfer procedure. () Gold(III) chloride trihydrate in water. () The addition of tetraoctylammonium bromide in toluene. (C) The addition of sodium borohydride in water. unps 1.Eu.Na Toluene unp- 1.Eu.Na Figure S9. Surface functionalisation and purification of unp-1.eu.na. () TO-stabilized unps functionalized with 1.Eu.Na using a modified rust-schiffrin twophase method. () Purification of unp-1.eu.na (purple band) by G15 Sephadex size exclusion column chromatography using Millipore H2O for the eluent.

10 Normalised bsorbance Intensity (a.u.) 1..9 unps unp-1.eu.na Figure S1. Photophysical characterization of unp-1.eu.na at 298 K. () bsorption spectra of TO-stabilized unps (in toluene) and the functionalized system unp-1.eu.na (in H2O). () Phosphorescence spectra (λexc = 395 nm) of unp-1.eu.na (1 1 7 M) recorded in H2O. Figure S11. TEM images of unp-1.eu.na (4 1 7 M) after deposition onto formvar stabilized carbon coated copper grids. () Large field view. () Close field view.

11 Figure S12. Zeta potential measurement for the system unp-1.eu.na. Figure S13. TEM images of unp-1.eu (4 1 7 M) after deposition onto formvar stabilized carbon coated copper grids. () Large field view. () Close field view.

12 Volume (%) Number of unps 3 v Core Diameter = (1 %) 4 35 v Core Diameter = 3.5 nm Diameter Size (nm) Figure S14. Particle size characterization of the functionalized gold nanoparticles by DLS and TEM. () Volume distribution profile determined by DLS analysis for unp-1.eu (1 1 7 M) in CH3OH at 298 K. () Size distribution profile calculated from analyzing the TEM images of unp-1.eu (4 1 7 M). Diameter of unps (nm) Figure S15. Zeta potential measurement for the system unp-1.eu.

13 bsorbance bsorbance bsorbance Intensity (a.u.) Intensity (a.u.) SUPPLEMENTL DT 1. Photophysical Measurements eq. nta 1.1 eq. nta 5.79 eq. nta eq. of nta Eq. of nta added Figure S16. Formation of a 1:1 ternary complex in solution, 1.Eu.Na-nta. () Phosphorescent response of 1.Eu.Na (1 1 5 M) upon titrating with nta ( eq.) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 33 nm). () Changes in the Eu(III)-centered emission as a function of nta equivalents, measured at 617 nm eq. nta 22 eq. nta 2 eq. nta Eq. of nta added Figure S17. bsorption studies showing the formation of 1:1 ternary complexes in solution on the surface of unps. () Changes in the absorbance of unp-1.eu.na (1 1 7 M) upon titrating with nta ( 2 eq.) in.1 M HEPES (I =.1) at ph 7.4 and 298 K. Inset: Magnified view. () Changes in the SPR band of unp-1.eu.na as a function of nta equivalents, measured at 525 nm.

14 Intensity (a.u.) Intensity (a.u.) Intensity (a.u.) Normalised Intensity eq. nta 22 eq. nta 2 eq. nta Figure S18. Phosphorescence studies showing the formation of 1:1 ternary complexes in solution on the surface of unps. () Phosphorescent response of 1.Eu.Na (1 1 5 M) upon titrating with nta ( eq.) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 33 nm). () Changes in the Eu(III)-centered emission as a function of nta equivalents, measured at 617 nm eq. of nta 21 2 eq. of nta Eq. of nta added D 2 O HEPES 6 5 eq. nta 22 eq. nta 2 eq. nta nm Figure S19. Fluorescence studies showing the formation of 1:1 ternary complexes in solution on the surface of unps. () Fluorescent response of unp-1.eu.na (1 1 7 M) upon titrating with nta ( 2 eq.) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 33 nm). () Changes in the antenna and Eu(III) emission as a function of nta equivalents, measured at 452 and 616 nm Eq. of nta added 452 nm

15 Intensity (a.u.) Integrated Emission bsorbance bsorbance.8 ph 8.76 ph ph Figure S2. bsorption studies showing the ph-dependence of unp-1.eu.na in aqueous solution. () Changes in the absorbance of unp-1.eu.na (1 1 7 M) as a function of ph in H2O (I =.1) at 298 K. () Changes in the SPR band of unp-1.eu.na as a function of ph, measured at 525 nm. ph ph 8.76 ph 7.1 ph Figure S21. Phosphorescence studies showing the ph-dependence of unp-1.eu.na in aqueous solution, basic acidic. () Phosphorescent response of unp-1.eu.na ( M) as a function of ph in H2O (I =.1) at 298 K (λexc = 395 nm). () Changes in the integrated Eu(III) emission as a function of ph, for both the forward (ph , red) and back (ph , blue) titration, measured between nm. ph

16 bsorbance bsorbance Intensity (a.u.) bsorbance ph 3.52 ph 7.5 ph Figure S22. Phosphorescence studies showing the ph-dependence of unp-1.eu.na in aqueous solution, acidic basic. () Phosphorescent response of unp-1.eu.na (1 1 7 M) as a function of ph, for the back titration (ph 4 9), in H2O (I =.1) at 298 K (λexc = 395 nm). () Changes in the SPR band of unp-1.eu.na (1 1 7 M) as a function of ph, for the back titration (ph 4 9), measured at 525 nm. ph.12.1 ph 8.75 ph 6.99 ph nm nm 33 nm 525 nm Figure S23. bsorption studies showing the ph-dependence of the ternary complex system unp-1.eu.na-nta in aqueous solution. () Changes in the absorption spectra of unp-1.eu.na (1 1 7 M) and nta (2 eq.) as a function of ph in H2O (I =.1) at 298 K. () Changes in the SPR band of unp-1.eu.na and the antenna nta as a function of ph, measured at 525, 282, 29, and 33 nm. ph

17 bsorbance bsorbance Intensity (a.u.) Intensity (a.u.) ph 8.75 ph 6.99 ph Figure S24. Fluorescence studies showing the ph-dependence of the ternary complex system unp-1.eu.na-nta in aqueous solution. () Fluorescent response of unp-1.eu.na (1 1 7 M) and nta (2 eq.) as a function of ph in H2O (I =.1) at 298 K (λexc = 33 nm). () Changes in the Eu(III) emission as a function of ph, for both the forward (ph , red) and back (ph , blue) titration, measured between nm. ph.3 ph 9.53 ph 7.6 ph nm nm 284 nm Figure S25. bsorption studies showing the ph-dependence of the 1:1 ternary complex 1.Eu.Na-nta in aqueous solution. () Changes in the absorption spectra of 1.Eu.Na-nta ( M) as a function of ph in H2O (I =.1) at 298 K. () Changes in the antenna nta as a function of ph, measured at 284, 291, and 331 nm. ph

18 Intensity (a.u.) Intensity (a.u.) bsorbance bsorbance.3 ph 9.53 ph 7.6 ph nm nm 284 nm Figure S26. Phosphorescence studies showing the ph-dependence of the 1:1 ternary complex 1.Eu.Na-nta in aqueous solution. () Phosphorescent response of 1.Eu.Na-nta ( M) as a function of ph in H2O (I =.1) at 298 K (λexc = 33 nm). () Changes in the integrated Eu(III) emission as a function of ph, for both the forward (ph , red) and back (ph , blue) titration, measured between nm. ph 6 ph 9.53 ph 7.6 ph Figure S27. Fluorescence studies showing the ph-dependence of the 1:1 ternary complex 1.Eu.Na-nta in aqueous solution. () Fluorescent response of 1.Eu.Na-nta ( M) as a function of ph in H2O (I =.1) at 298 K (λexc = 33 nm). () Changes in the Eu(III) emission as a function of ph, for both the forward (ph , red) and back (ph , blue) titration, measured at 616 nm. ph

19 Intensity (a.u.) bsorbance bsorbance.8.7 M CaCl M CaCl M CaCl x1-3 4.x1-3 6.x1-3 8.x1-3 1.x1-2 Conc. of CaCl 2 (M) Figure S28. bsorption studies showing the effect of Ca(II) on unp-1.eu.na. () Changes in the absorbance of unp-1.eu.na (1 1 7 M) upon titrating with CaCl2 (. 1. mm) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 395 nm). () Changes in the SPR band as a function of CaCl2, measured at 525 nm. 1 8 M CaCl M CaCl M CaCl I/I x1-3 4.x1-3 6.x1-3 8.x1-3 1.x1-2 Conc. of CaCl 2 (M) Figure S29. Phosphorescence studies showing the effect of Ca(II) on unp-1.eu.na. () Phosphorescent response of unp-1.eu.na (1 1 7 M) upon titrating with CaCl2 (. 1. mm) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 395 nm). () Changes in the integrated Eu(III) emission as a function of CaCl2, measured between nm.

20 bsorbance bsorbance Intensity (a.u.) M CaCl M CaCl M CaCl 2 (+12 h) I/I Time (hr) Figure S3. Phosphorescence studies showing the effect of Ca(II) on unp-1.eu.na after 12 hr. () Phosphorescent response of unp-1.eu.na (1 1 7 M) and CaCl2 (1 mm) as a function of time ( 12 hr) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 395 nm). () Changes in the integrated Eu(III) emission as a function of time, measured between nm..7.6 M Na 2 HPO M Na 2 HPO M Na 2 HPO x1-3 4.x1-3 6.x1-3 8.x1-3 1.x1-2 Conc. of Na 2 HPO 4 (M) Figure S31. bsorption studies showing the effect of PO4 3 on unp-1.eu.na. () Changes in the absorbance of unp-1.eu.na (1 1 7 M) upon titrating with disodium phosphate (. 1. mm) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 395 nm). () Changes in the SPR band as a function of disodium phosphate, measured at 525 nm.

21 Intensity (a.u.) Intensity (a.u) M Na 2 HPO M Na 2 HPO M Na 2 HPO I/I x1-3 4.x1-3 6.x1-3 8.x1-3 1.x1-2 Conc. of Na 2 HPO 4 (M) Figure S32. Phosphorescence studies showing the effect of PO4 3 on unp-1.eu.na. () Phosphorescent response of unp-1.eu.na (1 1 7 M) upon titrating with disodium phosphate (. 1. mm) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 395 nm). () Changes in the integrated Eu(III) emission as a function of disodium phosphate, measured between nm M Na 2 HPO M Na 2 HPO M Na 2 HPO 4 (+12 h) I/I Time (hr) Figure S33. Phosphorescence studies showing the effect of PO4 3 on unp-1.eu.na after 12 hr. () Phosphorescent response of unp-1.eu.na (1 1 7 M) and Na2HPO4 (1 mm) as a function of time ( 12 hr) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 395 nm). () Changes in the integrated Eu(III) emission as a function of time, measured between nm.

22 bsorbance bsorbance bsorbance Intensity (a.u.) Intensity (a.u.) eq. nta 1. eq. nta 4. eq. nta ±.1 eq. of nta Eq. of nta added Figure S34. Formation of a 1:1 ternary complex in solution, 1.Eu-nta. () Phosphorescent response of 1.Eu (1 1 5 M) upon titrating with nta (. 4. eq.) in CH3OH (TP =.1 M) at 298 K (λexc = 33 nm). () Changes in the Eu(III)-centered emission as a function of nta equivalents, measured at 617 nm eq. nta 1 eq. nta 5 eq. nta Eq. of nta added Figure S35. bsorption studies showing the formation of 1:1 ternary complexes in solution on the surface of unps. () Changes in the absorbance of unp-1.eu (1 1 7 M) upon titrating with nta ( 5 eq.) in CH3OH (TP =.1) at 298 K. Inset: Magnified view. () Changes in the SPR band of unp-1.eu as a function of nta equivalents, measured at 522 nm.

23 Intensity (a.u.) Intensity (a.u.) Intensity (a.u.) Intensity (a.u.) eq. nta 1 eq. nta 5 eq. nta eq. of nta Eq. of nta added Figure S36. Phosphorescence studies showing the formation of 1:1 ternary complexes in solution on the surface of unps. () The phosphorescent response of unp-1.eu (1 1 7 M) upon titrating with nta ( 5 eq.) in CH3OH (TP =.1 M) at 298 K (λexc = 33 nm). () Changes in the Eu(III)-centered emission as a function of nta equivalents, measured at 617 nm. 7 6 eq. nta 1 eq. nta 5 eq. nta nm nm Eq. of nta added Figure S37. Fluorescence studies showing the formation of 1:1 ternary complexes in solution on the surface of unps. () Fluorescent response of unp-1.eu (1 1 7 M) upon titrating with nta ( 5 eq.) in CH3OH (TP =.1 M) at 298 K (λexc = 33 nm). () Changes in the antenna and Eu(III) emission as a function of nta equivalents, measured at 48 and 616 nm.

24 Intensity (a.u.) bsorbance bsorbance.6.5 M TP M TP M TP x1-3 4.x1-3 6.x1-3 8.x1-3 1.x1-2 Conc. of TP (M) Figure S38. bsorption studies showing the effect of PO4 3 on unp-1.eu. () Changes in the absorbance of unp-1.eu (1 1 7 M) upon titrating with tetrabutylammonium phosphate (. 1. mm) in CH3OH at 298 K (λexc = 395 nm). () Changes in the SPR band as a function of tetrabutylammonium phosphate, measured at 525 nm M TP M TP M TP I/I x1-3 4.x1-3 6.x1-3 8.x1-3 1.x1-2 Conc. of TP (M) Figure S39. Phosphorescence studies showing the effect of PO4 3 on unp-1.eu. () Phosphorescent response of unp-1.eu (1 1 7 M) upon titrating with tetrabutylammonium phosphate (.. mm) in.1 M HEPES (I =.1) at ph 7.4 and 298 K (λexc = 395 nm). () Changes in the integrated Eu(III) emission as a function of tetrabutylammonium phosphate, measured between nm.

25 Intensity (cps) Intensity (cps) Intensity (a.u.) 14 M TP M TP M TP I/I Time (hr) Figure S4. Phosphorescence studies showing the effect of PO4 3 on unp-1.eu after 12 hr. () Phosphorescent response of unp-1.eu (1 1 7 M) and tetrabutylammonium phosphate (1 mm) as a function of time ( 12 hr) in CH3OH at 298 K (λexc = 395 nm). () Changes in the integrated Eu(III) emission as a function of time, measured between nm. 2. Solid-State Photophysical Measurements hr 4 hr hr Surface immersed Surface not immersed hr 4 hr hr Surface immersed Surface not immersed Figure S41. Phosphorescence studies of microdamaged bovine bone structure as a function of time immersed in unp-1.eu.na. () Concentration of unp-1.eu.na in.1 M HEPES (I =.1) at ph 7.4 = M; λexc = 395 nm. () Concentration of unp-1.eu.na in.1 M HEPES (I =.1) at ph 7.4 = M; λexc = 395 nm. Comparison between the surface of healthy bone being immersed and not immersed in unp-1.eu.na is also given.

26 Intensity (cps) Intensity (cps) Intensity (cps) Intensity (cps) hr 4 hr hr Surface immersed Surface not immersed hr 4 hr hr Surface immersed Surface not immersed Figure S42. Luminescence studies of microdamaged bovine bone structure after immersion in aqueous solution of unp-1.eu.na and nta. () Phosphorescent response (λexc = 33 nm) after immersing the same scratched bone specimen (already immersed in unp-1.eu.na) in nta ( M). () Fluorescent response (λexc = 33 nm) after immersing the same scratched bone specimen (already immersed in unp-1.eu.na) in nta ( M) hr 4 hr hr Surface immersed Surface not immersed hr 4 hr hr Surface immersed Surface not immersed Figure S43. Phosphorescence studies of microdamaged bovine bone structure as a function of time immersed in unp-1.eu. () Concentration of unp-1.eu in CH3OH = M; λexc = 395 nm. () Concentration of unp-1.eu in CH3OH = M; λexc = 395 nm. Comparison between the surface of healthy bone being immersed and not immersed in unp-1.eu is also given.

27 Intensity (cps) Intensity (cps) hr 4 hr hr Surface immersed Surface not immersed hr 4 hr hr Surface immersed Surface not immersed Figure S44. Luminescence studies of microdamaged bovine bone structure after immersion in a CH3OH solution of unp-1.eu and nta. () Phosphorescent response (λexc = 33 nm) after immersing the same scratched bone specimen (already immersed in unp-1.eu) in nta ( M). () Fluorescent response (λexc = 33 nm) after immersing the same scratched bone specimen (already immersed in unp-1.eu) in nta ( M).

28 3. Microscopy Studies hr 4 hr 24 hr Figure S45. Polarized light microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in an aqueous solution of unp-1.eu.na. () Concentration of unp-1.eu.na in.1 M HEPES (I =.1) at ph 7.4 = M. () Concentration of unp-1.eu.na in.1 M HEPES (I =.1) at ph 7.4 = M. ll scale bars = 25 μm. hr 4 hr 24 hr Figure S46. Epifluorescence microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in an aqueous solution of unp-1.eu.na, followed by immersion in an aqueous solution of nta ( M) for 3 sec. () Concentration of unp-1.eu.na in.1 M HEPES (I =.1) at ph 7.4 = M. () Concentration of unp-1.eu.na in.1 M HEPES (I =.1) at ph 7.4 = M. Each image was acquired using a LED source (λexc = 365 nm) and a 52 longpass emission filter; all scale bars = 25 μm.

29 24 hr 4 hr hr C Figure S47. TPE fluorescence microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in an aqueous solution of unp-1.eu.na ( M), followed by immersion in an aqueous solution of nta ( M) for 3 sec. () Images taken at the bottom of the microcrack. () Images taken at the top of the microcrack. (C) Whole projection images of the microcrack (z-stack). Each image was acquired using a Ti-Sapphire laser (λexc = 75 nm) and a red channel emission filter (λem = nm); all scale bars = 15 μm; aqueous solution = buffered.1 M HEPES (I =.1 M NaCl, ph 7.4).

30 24 hr 4 hr hr C Figure S48. TPE fluorescence microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in an aqueous solution of unp-1.eu.na ( M), followed by immersion in an aqueous solution of nta ( M) for 3 sec. () Images taken at the bottom of the microcrack. () Images taken at the top of the microcrack. (C) Whole projection images of the microcrack (z-stack). Each image was acquired using a Ti-Sapphire laser (λexc = 75 nm) and a red channel emission filter (λem = nm); all scale bars = 15 μm; aqueous solution = buffered.1 M HEPES (I =.1 M NaCl, ph 7.4).

31 C hr 4 hr 24 hr Figure S49. Polarized light microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in a CH3OH solution of unp-1.eu. () Concentration of unp-1.eu = M. () Concentration of unp-1.eu = M. (C) Concentration of unp-1.eu = M. ll scale bars = 25 μm.

32 C hr 4 hr 24 hr Figure S5. Epifluorescence microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in a CH3OH solution of unp-1.eu, followed by immersion in a CH3OH solution of nta ( M) for 3 sec. () Concentration of unp-1.eu = M. () Concentration of unp-1.eu = M. (C) Concentration of unp-1.eu = M. Each image was acquired using a LED source (λexc = 365 nm) and a 52 longpass emission filter; all scale bars = 25 μm.

33 24 hr 4 hr hr C Figure S51. TPE fluorescence microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in a CH3OH solution of unp-1.eu ( M), followed by immersion in a CH3OH solution of nta ( M) for 3 sec. () Images taken at the bottom of the microcrack. () Images taken at the top of the microcrack. (C) Whole projection images of the microcrack (z-stack). Each image was acquired using a Ti-Sapphire laser (λexc = 75 nm) and a red channel emission filter (λem = nm); all scale bars = 15 μm.

34 24 hr 4 hr hr C Figure S52. TPE fluorescence microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in a CH3OH solution of unp-1.eu ( M), followed by immersion in a CH3OH solution of nta ( M) for 3 sec. () Images taken at the bottom of the microcrack. () Images taken at the top of the microcrack. (C) Whole projection images of the microcrack (z-stack). Each image was acquired using a Ti-Sapphire laser (λexc = 75 nm) and a red channel emission filter (λem = nm); all scale bars = 15 μm.

35 24 hr 4 hr hr C Figure S53. TPE fluorescence microscopy images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in a CH3OH solution of unp-1.eu ( M), followed by immersion in a CH3OH solution of nta ( M) for 3 sec. () Images taken at the bottom of the microcrack. () Images taken at the top of the microcrack. (C) Whole projection images of the microcrack (z-stack). Each image was acquired using a Ti-Sapphire laser (λexc = 75 nm) and a red channel emission filter (λem = nm); all scale bars = 15 μm.

36 24 hr 4 hr hr 24 hr 4 hr hr Figure S54. TPE microscopy 2D snapshot images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in a CH3OH solution of unp-1.eu, followed by immersion in a CH3OH solution of nta ( M) for 3 sec. () Concentration of unp-1.eu = M. () Concentration of unp-1.eu = M. Figure S55. TPE microscopy 3D snapshot images of microdamaged bovine bone structure after being immersed for, 4, and 24 hr in a CH3OH solution of unp-1.eu, followed by immersion in a CH3OH solution of nta ( M) for 3 sec. () Concentration of unp-1.eu = M. () Concentration of unp-1.eu = M.

37 Intensity (a.u.) Intensity (a.u.) Figure S56. Phosphorescence spectra obtained from quantum yield determination. () 1.Eu.Na ( M). () unp-1.eu.na ( M). Supplemental References 1. McMahon,., Mauer, P., McCoy, C.P., Lee, T.C., and Gunnlaugsson, T. (29). Selective imaging of damaged bone structure (microcracks) using a targeting supramolecular Eu(III) complex as a lanthanide luminescent contrast agent. J. m. Chem. Soc. 131, McMahon,. K., and Gunnlaugsson, T. (21). Lanthanide luminescence sensing of copper and mercury ions using an iminodiacetate-based Tb(III)-cyclen chemosensor. Tetrahedron Lett. 51, Comby, S., and Gunnlaugsson, T. (211). Luminescent lanthanide-functionalized gold nanoparticles: exploiting the interaction with bovine serum albumin for potential sensing applications. CS. Nano. 5, Truman, L.K., Comby, S., and Gunnlaugsson, T. (212). ph-responsive luminescent lanthanide-functionalized gold nanoparticles with "on-off" ytterbium switchable nearinfrared emission. ngew. Chem. Int. Ed. 51, Massue, J., Quinn, S.J., and Gunnlaugsson, T. (28). Lanthanide luminescent displacement assays: the sensing of phosphate anions using Eu(III)-cyclen-conjugated gold nanoparticles in aqueous solution. J. m. Chem. Soc. 13, Yokokawa, S., Tamada, K., Ito, E., and Hara, M. (23). Cationic self-assembled monolayers composed of gemini-structured dithiol on gold: new concept for molecular recognition because of the distance between adsorption sites. J. Phys. Chem.. 17, Massue, J., Plush, S.E., onnet, C.S., Moore, D.., and Gunnlaugsson, T. (27). Selective mono N-alkylations of cyclen in one step syntheses. Tett. Lett. 48,