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Chem, Volume 1

Supplemental Information

Two-Photon Luminescent Bone Imaging

Using Europium Nanoagents

Esther M. Surender, Steve Comby, Brenton L. Cavanagh, Orlaith Brennan, T. CliveLee, and Thorfinnur Gunnlaugsson

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

1. General Experimental Procedures

Procedure 1: Synthesis of Ln(III) complexes using Ln(CF3SO3)3

All Ln(III) complexes found within were prepared by refluxing, under microwave irradiation, the

relevant ligand with 1.0 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 (200 mL). Owing to the paramagnetic nature of the Ln(III) ion, 1H NMR

spectra of the complexes consisted of broad resonances and, therefore, were not fully

characterized in terms of integration. These paramagnetic properties also prevented 13C NMR

spectra from being recorded.

Procedure 2: Base hydrolysis of the diethyliminodiacetate functional groups1,2

Alkaline hydrolysis was carried out by refluxing, under microwave irradiation, the relevant Ln(III)

complex with 6.0 eq. of NaOH for 24 hr in a mixed CH3OH/H2O (1:9 v/v) solution. After 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 IPA. 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 6.0 eq of aqueous NaOH.

Procedure 3: Gold nanoparticle synthesis3,4

Au(III) chloride trihydrate (0.10 g, 0.25 mmol) was dissolved in Millipore H2O (10 mL) and

tetraoctylammonium bromide (0.36 g, 0.26 mmol) in toluene (25 mL). The two solutions were

then stirred vigorously at room temperature for 10 min. NaBH4 (0.12 g, 3.17 mmol) dissolved in

Millipore H2O (10 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 Au(0) into the toluene layer, the organic and aqueous layers were separated,

and the toluene layer was washed with H2O (2 × 20 mL), 0.1 M HCl (2 × 20 mL), and 0.1 NaOH

(2 × 20 mL). Formation of the TOAB-stabilized AuNPs was confirmed by UV-vis absorption

spectroscopy by the SPR band observed at 525 nm in toluene.3-5

Procedure 4: Surface functionalization of AuNPs with Ln(III) complexes3,4

AuNP functionalization was carried out by vigorously stirring a solution of the TOAB-stabilized

AuNPs (5 mL) and the relevant aqueous Ln(III) solution (2.76 mM, 5 mL) in the presence of

NaBH4 (105.74 mM, 500 μL) at room temperature for 16 hr. Following successful transfer of the

AuNPs into the aqueous layer, the two layers were separated, and the aqueous layer was

filtered through a PDVF 0.45 μm microsyringe to give a clear purple solution. Any unbound

Ln(III) complex was then removed by either sephadex G-15 or LH-20 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 TOAB-stabilized AuNPs.3-5

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-Bromoundecane-1-thiol (2)6

Acetyl chloride (6.34 g, 80.77 mmol) was added slowly to a stirred 0 °C CH3OH

(40 mL) solution. S-(11-bromoundecyl)thioacetate (1.00 g, 0.32 mmol) was then

added slowly along with CH3OH (10 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 × 30 mL), and extracted with CH2Cl2 (2 × 30 mL). The combined

organic layers were dried over MgSO4, filtered, and the solvent removed under reduced

pressure to yield 2 as a white solid (0.80 g, 3.01 mmol, 93% yield). HRMS (m/z, MALDI):

Calculated for C11H23SBr m/z = 530.1251 [2M−2H]+. Found m/z = 530.1266; 1H NMR (400 MHz,

CDCl3) δH: 3.40 (2H, t, J = 6.89, CH2Br), 2.52 (2H, q, J = 7.20, CH2SH), 1.85 (2H, qu, J = 7.20,

CH2CH2Br), 1.60 (2H, qu, J = 7.50, CH2CH2SH), 1.44 – 1.27 (14H, m, 7 × CH2); 13C NMR (400 MHz,

CDCl3) δC: 34.24 (CH2), 34.17 (CH2), 32.95 (CH2), 29.64 (CH2), 29.62 (CH2), 29.55 (CH2), 29.19

(CH2), 28.89 (CH2), 28.49 (CH2), 28.29 (CH2), 24.79 (CH2); IR υmax (cm−1): 2925, 2859, 1460, 1261,

1099, 1029, 905, 804, 731.

1,2-Bis-(11-bromoundecyl)disulfane (3)6

Compound 2 (0.60 g, 2.27 mmol) was dissolved along with iodine (0.17

g, 0.68 mmol) in CH2Cl2 (50 mL), and the resulting red solution was

refluxed for 16 hr. After reaction completion, the organic solution was washed with H2O (3 × 30

mL), dried over MgSO4, filtered, and the solvent removed under reduced pressure. The

product, 3, was obtained as a clear viscous oil (0.57 g, 1.08 mmol, 94% yield). HRMS (m/z,

MALDI): Calculated for C22H44S2Br2 m/z = 530.1251 [M]+. Found m/z = 530.1284; 1H NMR (400

MHz, CDCl3) δH: 3.41 (4H, t, J = 6.91, 2 × CH2Br), 2.68 (4H, t, J = 7.41, 2 × CH2S), 1.85 (4H, qu, J =

7.29, 2 × CH2CH2Br), 1.60 (4H, qu, J = 7.50, 2 × CH2CH2S), 1.44 – 1.27 (28H, m, 14 × CH2); 13C NMR

(400 MHz, CDCl3) δC: 39.35 (CH2), 34.19 (CH2), 32.99 (CH2), 29.67 (CH2), 29.65 (CH2), 29.62 (CH2),

29.57 (CH2), 29.37 (CH2), 28.91 (CH2), 28.66 (CH2), 28.32 (CH2); IR υmax (cm−1): 2916, 2850, 1463,

1280, 1225, 1019, 958, 885, 717, 640.

1,2-Bis[11-(1,4,7,10-tetraazacyclododecan-1-yl)undecyl]disulfane (4)7

Cyclen (1.19 g, 6.91 mmol) was dissolved along with NEt3

(0.21 g, 2.08 mmol) in freshly distilled CHCl3 (50 mL). After

stirring the solution for 5 min, 3 (0.46 g, 0.82 mmol) was

added and the resulting solution was refluxed for 16 hr

under an inert atmosphere. After cooling to room

temperature, the organic solution was washed with 1 M

NaOH (3 × 20 mL) to remove the excess cyclen and with

H2O (3 × 10 mL). The organic layer was dried over MgSO4, filtered, and the solvent removed

under reduced pressure to yield the product 4 (0.42 g, 0.59 mmol, 72% yield) as a yellow oil.

HRMS (m/z, ESI+): Calculated for C38H83N8S2 m/z = 715.6182 [M+H]+. Found m/z = 715.6183; 1H

NMR (400 MHz, CDCl3) δH: 2.81 – 2.50 (32H, m, cyclen-CH2), 2.38 (4H, t, J = 7.26, NCH2), 1.63 (4H,

q, J = 7.40, CH2CH2S), 1.45 – 1.24 (36H, m, 18 × CH2); 13C NMR (400 MHz, CDCl3) δC: 54.86 (CH2),

51.71 (CH2), 47.34 (CH2), 46.25 (CH2), 45.54 (CH2), 39.34 (CH2), 29.79 (CH2), 29.71 (CH2), 29.6 9

(CH2), 29.39 (CH2), 28.70 (CH2), 27.72 (CH2), 27.63 (CH2), 22.43 (CH2); IR υmax (cm−1): 2920, 2850,

1669, 1456, 1351, 1272, 1112, 1050, 935, 727, 601.

Diethyl 2,2-(2-chloroacetylamino)diacetate (5)7

Diethyl iminodiacetate (3.00 g, 15.86 mmol) and NEt3 (2.55 g, 25.20 mmol)

were dissolved in CH2Cl2 (50 mL). The clear solution was cooled to 0 °C and

chloroacetyl chloride (3.54 g, 31.34 mmol) was added slowly along with

CH2Cl2 (30 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 × 30 mL), 0.1 M HCl (2 × 30 mL), and brine (2 × 30

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, 14.57 mmol, 92% yield). HRMS (m/z, ESI+):

Calculated for C10H16NO5NaCl m/z = 288.0615 [M+Na]+. Found m/z = 288.0611; 1H NMR (400

MHz, CDCl3) δH: 4.22 (8H, m, CO2CH2CH3), 4.12 (2H, s, COCH2Cl), 1.30 (3H, t, J = 7.20, CH2CH3),

1.27 (3H, t, J = 7.20, CH2CH3); 13C NMR (400 MHz, CDCl3) δC: 168.64 (qt), 168.38 (qt), 167.87 (qt),

62.14 (CH2), 61.68 (CH2), 50.48 (CH2), 48.71 (CH2), 40.60 (CH2), 14.10 (CH3), 14.08 (CH3); IR υmax

(cm−1): 2986, 1738, 1661, 1463, 1407, 1375, 1299, 1260, 1188, 1063, 966, 931, 866, 796, 736.

1,4,7-Tris-(N,N-bis(ethoxycarbonylmethyl)-10-[dodecane-1-thiol]-1,4,7,10 tetraazacyclodo-

decane (1)

Ligand 4 (0.20 g, 0.28 mmol) was dissolved in freshly distilled

CH3CN (20 mL) in the presence of Cs2CO3 (0.60 g, 1.84 mmol)

and KI (0.31 g, 1.87 mmol). Compound 5 (0.47 g, 1.77 mmol)

was then added and the resulting solution was exposed to

microwave irradiation for 24 hr at 85 °C with 30 sec pre-stirring.

After cooling, removal of the inorganic salts was achieved by

centrifugation and the solvent was then reduced to ca. 10 mL.

Following NaBH4 (0.021 g, 0.56 mmol) was added, and the

solution was stirred at room temperature for 5 hr under an inert

atmosphere. After 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 100 to 80:20

CH2Cl2:CH3OH. The desired product, 1, was obtained as a viscous orange oil (0.21 g, 0.20

mmol, 36% yield). HRMS (m/z, ESI+): Calculated for C98H174N14O30S2 m/z = 1045.5975 [2M+2H]2+.

Found m/z = 1045.5985; 1H NMR (600 MHz, CDCl3) δH: 4.30 – 4.02 (24H, m, (CH2CO2CH2CH3)2),

3.72 – 2.61 (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); 13C NMR (600 MHz, CDCl3) δC: 171.75 (qt), 171.68

(qt), 171.39 (qt), 171.08 (qt), 170.93 (qt), 169.09 (qt), 168.93 (qt), 168.77 (qt), 61.99 (CH2), 61.92

(CH2), 61.34 (CH2), 60.38 (CH2), 60.08 (CH2), 53.51 (CH2), 52.69 (CH2), 52.40 (CH2), 50.49 (CH2),

50.09 (CH2), 48.54 (CH2), 48.09 (CH2), 39.09 (CH2), 29.48 (CH2), 29.20 (CH2), 28.46 (CH2), 27.47

(CH2), 22.62 (CH2), 21.03 (CH2), 14.18 (CH3); Calculated for C49H87N7O15S.1CHCl3.1CH2Cl2.1H2O:

C, 48.28; H, 7.31; N, 7.73. Found C, 48.29; H, 7.23; N, 7.80; IR υmax (cm−1): 2924, 2855, 1736, 1671,

1597, 1558, 1457, 1406, 1369, 1303, 1190, 1099, 1023, 876, 747, 660.

Complex 1.Eu

Complex Eu.1 was synthesised according to Procedure 1 using

ligand 1 (0.15 g, 0.14 mmol) and Eu(CF3SO3)3 (0.094 g, 0.16

mmol) . A viscous yellow oil was obtained (0.19 g, 0.12 mmol,

79% yield). HRMS (m/z, MALDI): Calculated for

C50H87N7O18F3S2Eu m/z = 1347.4714 [M+CF3SO3]+. Found m/z =

1347.4675; 1H NMR (400 MHz, CD3OD) δH: 33.86, 26.06, 23.81,

21.76, 20.40, 19.62, 19.03, 17.76, 17.18, 12.20, 4.93, 4.28, 3.78,

3.38, 3.35, 2.73, 1.71, 1.37, −0.39, −5.63, −6.64, −7.61, −8.98,

−9.86, −10.41, −11.32, −12.39, −14.44, −15.32, −16.49, −19.13,

−19.81; IR υmax (cm−1): 2929, 2860, 1742, 1612, 1535, 1457, 1375,

1224, 1163, 1081, 1023, 840, 725, 628.

Complex 1.Eu.Na

Complex Eu.1.Na was synthesised according to Procedure 2

using Eu.1 (0.19 g, 0.11 mmol) and NaOH (0.028 g, 0.70 mmol).

The desired product was obtained as a pale yellow solid (0.18

g, 0.12 mmol, 69% yield). m.p. decomposed above 260 °C;

HRMS (m/z, MALDI): Calculated for C37H61N7O15Eu m/z =

1028.3159 [M−6Na+4H]+. Found m/z = 1028.3204; 1H NMR (400

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, 0.64, −0.60, −0.77; IR υmax (cm−1): 2922, 2859,

1606, 1540, 1507, 1401, 1236, 1078, 1018, 978, 842, 709, 631.

3. Characterization

Figure S1. 1H NMR (600 MHz) of ligand 1 in CDCl3.

Figure S2. 13C NMR (600 MHz) of ligand 1 in CDCl3.

Figure S3. 1H NMR (400 MHz) of complex 1.Eu in CD3OD.

Figure S4. The HRMS isotopic distribution pattern for complex 1.Eu.

(A) Calculated pattern.

(B) Observed pattern.

1345 1346 1347 1348 1349 1350 1351 1352

0

20

40

60

80

100

%

m/z

Experimental

Calculated

1345 1346 1347 1348 1349 1350 1351 1352

0

20

40

60

80

100

13

50

.48

83

13

51

.47

58

13

45

.47

09

1346.4

727

13

47

.46

86

13

49

.48

17

13

51

.50

13

13

48

.47

33

13

50

.47

51

1346.4

729

13

49

.47

42

13

48

.47

42

13

47

.47

16

13

45

.46

98

%

A

B

Figure S5. 1H NMR (400 MHz) of complex 1.Eu.Na in D2O.

Figure S6. The HRMS isotopic distribution pattern for complex 1.Eu.Na.

(A) Calculated pattern.

(B) Observed pattern.

1026 1027 1028 1029 1030 1031 1032 1033

0

20

40

60

80

100

%

m/z

Experimental

Calculated

1026 1027 1028 1029 1030 1031 1032 1033

0

20

40

60

80

100

10

31

.31

21

1032.3

215

1026.3

175

1027.3

240

1028.3

204

1030.3

137

1032.3

293

1029.3

276

1031.3

201

1027.3

14

1030.3

190

1029.3

188

1028.3

160

1026.3

144

%

A

B

Figure S7. Luminescence lifetime decay of 1.Eu.Na fitted with a mono-exponential function.

(A) Measured in H2O.

(B) Measured in D2O.

Figure S8. Reduction of Au(III) to Au(0) using a modified Brust-Schiffrin two-phase transfer

procedure.

(A) Gold(III) chloride trihydrate in water.

(B) The addition of tetraoctylammonium bromide in toluene.

(C) The addition of sodium borohydride in water.

Figure S9. Surface functionalisation and purification of AuNP-1.Eu.Na.

(A) TOAB-stabilized AuNPs functionalized with 1.Eu.Na using a modified Brust-Schiffrin two-

phase method.

(B) Purification of AuNP-1.Eu.Na (purple band) by G15 Sephadex size exclusion column

chromatography using Millipore H2O for the eluent.

0 2 4 6 8 10 12 14

0

100

200

300

400

500In

ten

sity

(a

.u.)

Time (ms)

0 2 4 6 8 10 12 14

0

100

200

300

400

500

Inte

nsi

ty (

a.u

.)

Time (ms)

AuNP-

1.Eu.Na

Toluene

1.Eu.Na

AuNPs

A B

A B C

A

B

Figure S10. Photophysical characterization of AuNP-1.Eu.Na at 298 K.

(A) Absorption spectra of TOAB-stabilized AuNPs (in toluene) and the functionalized system

AuNP-1.Eu.Na (in H2O).

(B) Phosphorescence spectra (λexc = 395 nm) of AuNP-1.Eu.Na (1 × 10−7 M) recorded in H2O.

Figure S11. TEM images of AuNP-1.Eu.Na (4 × 10−7 M) after deposition onto formvar stabilized

carbon coated copper grids.

(A) Large field view.

(B) Close field view.

580 600 620 640 660 680 700 720

0

20

40

60

80

100

Inte

nsi

ty(a

.u.)

Wavelength (nm)

300 350 400 450 500 550 600 650 700

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0N

orm

ali

sed

Ab

sorb

an

ce

Wavelength (nm)

AuNPs

AuNP-1.Eu.Na

A B

A B

Figure S12. Zeta potential measurement for the system AuNP-1.Eu.Na.

Figure S13. TEM images of AuNP-1.Eu (4 × 10−7 M) after deposition onto formvar stabilized

carbon coated copper grids.

(A) Large field view.

(B) Close field view.

A B

Figure S14. Particle size characterization of the functionalized gold nanoparticles by DLS and

TEM.

(A) Volume distribution profile determined by DLS analysis for AuNP-1.Eu (1 × 10−7 M) in CH3OH

at 298 K.

(B) Size distribution profile calculated from analyzing the TEM images of AuNP-1.Eu (4 × 10−7 M).

Figure S15. Zeta potential measurement for the system AuNP-1.Eu.

1 2 3 4 5 6 7 8

0

5

10

15

20

25

30

35

40Av Core Diameter = 3.5 nm

Nu

mb

er o

f A

uN

Ps

Diameter of AuNPs (nm)

1 10 100 1000 10000

0

5

10

15

20

25

30Av Core Diameter = 15.57 (100 %)

Vo

lum

e (%

)

Diameter Size (nm)

A B

SUPPLEMENTAL DATA

1. Photophysical Measurements

Figure S16. Formation of a 1:1 ternary complex in solution, 1.Eu.Na-nta.

(A) Phosphorescent response of 1.Eu.Na (1 × 10−5 M) upon titrating with nta (0.00→5.79 eq.) in

0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K (λexc = 330 nm).

(B) Changes in the Eu(III)-centered emission as a function of nta equivalents, measured at

617 nm.

Figure S17. Absorption studies showing the formation of 1:1 ternary complexes in solution on

the surface of AuNPs.

(A) Changes in the absorbance of AuNP-1.Eu.Na (1 × 10−7 M) upon titrating with nta (0→200

eq.) in 0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K. Inset: Magnified view.

(B) Changes in the SPR band of AuNP-1.Eu.Na as a function of nta equivalents, measured at

525 nm.

580 600 620 640 660 680 700 720

0

40

80

120

160

200

240

Inte

nsi

ty(a

.u.)

Wavelength (nm)

0.00 eq. nta

1.01 eq. nta

5.79 eq. nta

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

0

50

100

150

200

250

1 0.1 eq. of nta

Inte

nsi

ty(a

.u.)

Eq. of nta added

400 450 500 550 600 650 700

0.000

0.008

0.016

0.024

0.032

0.040

Ab

sorb

an

ce

Wavelength (nm)

250 300 350 400 450 500 550 600 650 700

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Ab

sorb

an

ce

Wavelength (nm)

0 eq. nta

22 eq. nta

200 eq. nta

0 25 50 75 100 125 150 175 200

0.0225

0.0250

0.0275

0.0300

0.0325

0.0350

0.0375

0.0400

Ab

sorb

an

ce

Eq. of nta added

A B

A B

Figure S18. Phosphorescence studies showing the formation of 1:1 ternary complexes in

solution on the surface of AuNPs.

(A) Phosphorescent response of 1.Eu.Na (1 × 10−5 M) upon titrating with nta (0.00→5.79 eq.) in



617 nm.

Figure S19. Fluorescence studies showing the formation of 1:1 ternary complexes in solution on


(A) Fluorescent response of AuNP-1.Eu.Na (1 × 10−7 M) upon titrating with nta (0→200 eq.) in


(B) Changes in the antenna and Eu(III) emission as a function of nta equivalents, measured at

452 and 616 nm.

0 10 20 30 40 50 60 70 80 90 100 110 120

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

18 2 eq. of nta

21 2 eq. of nta

HEPES

D2O

No

rma

lise

dIn

ten

sity

Eq. of nta added

580 600 620 640 660 680 700 720

0

100

200

300

400

500

600

700

8000 eq. nta

22 eq. nta

200 eq. nta

Inte

nsi

ty(a

.u.)

Wavelength (nm)

400 450 500 550 600 650 700

0

100

200

300

400

500

6000 eq. nta

22 eq. nta

200 eq. nta

Inte

nsi

ty(a

.u.)

Wavelength (nm)

0 25 50 75 100 125 150 175 200

0

10

20

30

40

50

200

300

400

500

452 nm

616 nm

Inte

nsi

ty(a

.u.)

Eq. of nta added

A B

A B

Figure S20. Absorption studies showing the pH-dependence of AuNP-1.Eu.Na in aqueous

solution.

(A) Changes in the absorbance of AuNP-1.Eu.Na (1 × 10−7 M) as a function of pH in H2O (I =

0.1) at 298 K.

(B) Changes in the SPR band of AuNP-1.Eu.Na as a function of pH, measured at 525 nm.

Figure S21. Phosphorescence studies showing the pH-dependence of AuNP-1.Eu.Na in

aqueous solution, basic→acidic.

(A) Phosphorescent response of AuNP-1.Eu.Na (1.0 × 10−7 M) as a function of pH in H2O (I = 0.1)

at 298 K (λexc = 395 nm).

(B) Changes in the integrated Eu(III) emission as a function of pH, for both the forward (pH

9.0→3.5, red) and back (pH 3.5→9.0, blue) titration, measured between 570–720 nm.

250 300 350 400 450 500 550 600 650 700

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08 pH 8.76

pH 7.01

pH 3.52

Ab

sorb

an

ce

Wavelength (nm)

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Ab

sorb

an

ce

pH

580 600 620 640 660 680 700 720

0

10

20

30

40

50

pH 8.76

pH 7.01

pH 3.52

Inte

nsi

ty(a

.u.)

Wavelength (nm)

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

0

300

600

900

1200

1500

1800

Inte

gra

ted

Em

issi

on

pH

A B

A B

Figure S22. Phosphorescence studies showing the pH-dependence of AuNP-1.Eu.Na in

aqueous solution, acidic→basic.

(A) Phosphorescent response of AuNP-1.Eu.Na (1 × 10−7 M) as a function of pH, for the back

titration (pH 4→9), in H2O (I = 0.1) at 298 K (λexc = 395 nm).

(B) Changes in the SPR band of AuNP-1.Eu.Na (1 × 10−7 M) as a function of pH, for the back

titration (pH 4→9), measured at 525 nm.

Figure S23. Absorption studies showing the pH-dependence of the ternary complex system

AuNP-1.Eu.Na-nta in aqueous solution.

(A) Changes in the absorption spectra of AuNP-1.Eu.Na (1 × 10−7 M) and nta (20 eq.) as a

function of pH in H2O (I = 0.1) at 298 K.

(B) Changes in the SPR band of AuNP-1.Eu.Na and the antenna nta as a function of pH,

measured at 525, 282, 290, and 330 nm.

580 600 620 640 660 680 700 720

0

5

10

15

20

25

30

pH 3.52

pH 7.05

pH 8.94

Inte

nsi

ty(a

.u.)

Wavelength (nm)

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Ab

sorb

an

ce

pH

250 300 350 400 450 500 550 600 650 700

0.00

0.02

0.04

0.06

0.08

0.10

0.12pH 8.75

pH 6.99

pH 3.51

Ab

sorb

an

ce

Wavelength (nm)

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

282 nm

330 nm

525 nm

290 nm

Ab

sorb

an

ce

pH

A B

A B

Figure S24. Fluorescence studies showing the pH-dependence of the ternary complex system

AuNP-1.Eu.Na-nta in aqueous solution.

(A) Fluorescent response of AuNP-1.Eu.Na (1 × 10−7 M) and nta (20 eq.) as a function of pH in

H2O (I = 0.1) at 298 K (λexc = 330 nm).

(B) Changes in the Eu(III) emission as a function of pH, for both the forward (pH 9.0→3.5, red)

and back (pH 3.5→9.0, blue) titration, measured between 570 – 720 nm.

Figure S25. Absorption studies showing the pH-dependence of the 1:1 ternary complex

1.Eu.Na-nta in aqueous solution.

(A) Changes in the absorption spectra of 1.Eu.Na-nta (2.5 × 10−5 M) as a function of pH in H2O

(I = 0.1) at 298 K.

(B) Changes in the antenna nta as a function of pH, measured at 284, 291, and 331 nm.

400 450 500 550 600 650 700

0

75

150

225

300

375

450pH 8.75

pH 6.99

pH 3.51

Inte

nsi

ty(a

.u.)

Wavelength (nm)

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

0

50

100

150

200

250

300

Inte

nsi

ty(a

.u.)

pH

250 275 300 325 350 375 400 425 450 475 500

0.00

0.05

0.10

0.15

0.20

0.25

0.30 pH 9.53pH 7.06pH 3.50

Ab

sorb

an

ce

Wavelength (nm)

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

0.00

0.04

0.08

0.12

0.16

0.20

331 nm

284 nm

291 nm

Ab

sorb

an

ce

pH

A B

A B

Figure S26. Phosphorescence studies showing the pH-dependence of the 1:1 ternary complex


(A) Phosphorescent response of 1.Eu.Na-nta (2.5 × 10−5 M) as a function of pH in H2O (I = 0.1) at

298 K (λexc = 330 nm).

(B) Changes in the integrated Eu(III) emission as a function of pH, for both the forward (pH

9.5→3.5, red) and back (pH 3.5→9.0, blue) titration, measured between 570 – 720 nm.

Figure S27. Fluorescence studies showing the pH-dependence of the 1:1 ternary complex


(A) Fluorescent response of 1.Eu.Na-nta (2.5 × 10−5 M) as a function of pH in H2O (I = 0.1) at

298 K (λexc = 330 nm).

(B) Changes in the Eu(III) emission as a function of pH, for both the forward (pH 9.5→3.5, red)

and back (pH 3.5→9.0, blue) titration, measured at 616 nm.

250 275 300 325 350 375 400 425 450 475 500

0.00

0.05

0.10

0.15

0.20

0.25

0.30 pH 9.53pH 7.06pH 3.50

Ab

sorb

an

ce

Wavelength (nm)

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

0.00

0.04

0.08

0.12

0.16

0.20

331 nm

284 nm

291 nm

Ab

sorb

an

ce

pH

400 450 500 550 600 650 700

0

100

200

300

400

500

600 pH 9.53pH 7.06pH 3.50

Inte

nsi

ty (

a.u

.)

Wavelength (nm)

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

0

100

200

300

400

500

600

700

Inte

nsi

ty (

a.u

.)

pH

A B

A B

Figure S28. Absorption studies showing the effect of Ca(II) on AuNP-1.Eu.Na.

(A) Changes in the absorbance of AuNP-1.Eu.Na (1 × 10−7 M) upon titrating with CaCl2

(0.0→10.0 mM) in 0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K (λexc = 395 nm).

(B) Changes in the SPR band as a function of CaCl2, measured at 525 nm.

Figure S29. Phosphorescence studies showing the effect of Ca(II) on AuNP-1.Eu.Na.

(A) Phosphorescent response of AuNP-1.Eu.Na (1 × 10−7 M) upon titrating with CaCl2 (0.0→10.0

mM) in 0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K (λexc = 395 nm).

(B) Changes in the integrated Eu(III) emission as a function of CaCl2, measured between 570 –

720 nm.

0.0 2.0x10-3

4.0x10-3

6.0x10-3

8.0x10-3

1.0x10-2

0.026

0.028

0.030

0.032

0.034

0.036

0.038

Ab

sorb

an

ce

Conc. of CaCl2 (M)

250 300 350 400 450 500 550 600 650 700

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Ab

sorb

an

ce

Wavelength (nm)

0 M CaCl2

1 × 10-3 M CaCl2

1 × 10-3 M CaCl2

0.0 2.0x10-3

4.0x10-3

6.0x10-3

8.0x10-3

1.0x10-2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

I/I 0

Conc. of CaCl2 (M)

580 600 620 640 660 680 700 720

0

20

40

60

80

100

Inte

nsi

ty(a

.u.)

Wavelength (nm)

0 M CaCl2

1 × 10-3 M CaCl2

1 × 10-2 M CaCl2

A B

A B

Figure S30. Phosphorescence studies showing the effect of Ca(II) on AuNP-1.Eu.Na after 12 hr.

(A) Phosphorescent response of AuNP-1.Eu.Na (1 × 10−7 M) and CaCl2 (10 mM) as a function of

time (0→12 hr) in 0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K (λexc = 395 nm).

(B) Changes in the integrated Eu(III) emission as a function of time, measured between 570 –

720 nm.

Figure S31. Absorption studies showing the effect of PO43− on AuNP-1.Eu.Na.

(A) Changes in the absorbance of AuNP-1.Eu.Na (1 × 10−7 M) upon titrating with disodium

phosphate (0.0→10.0 mM) in 0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K (λexc = 395 nm).

(B) Changes in the SPR band as a function of disodium phosphate, measured at 525 nm.

580 600 620 640 660 680 700 720

0

10

20

30

40

50

60

70

80

90

100

Inte

nsi

ty(a

.u.)

Wavelength (nm)

0 M CaCl2

1 × 10-2 M CaCl2

1 × 10-2 M CaCl2 (+12 h)

0 1 2 3 4 5 6 7 8 9 10 11 12

0.0

0.2

0.4

0.6

0.8

1.0

1.2

I/I 0

Time (hr)

250 300 350 400 450 500 550 600 650 700

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Ab

sorb

an

ce

Wavelength (nm)

0 M Na2HPO4

1 × 10-3 M Na2HPO4

1 × 10-2 M Na2HPO4

0.0 2.0x10-3

4.0x10-3

6.0x10-3

8.0x10-3

1.0x10-2

0.015

0.020

0.025

0.030

0.035

0.040

Ab

sorb

an

ce

Conc. of Na2HPO4 (M)

A B

A B

Figure S32. Phosphorescence studies showing the effect of PO43− on AuNP-1.Eu.Na.

(A) Phosphorescent response of AuNP-1.Eu.Na (1 × 10−7 M) upon titrating with disodium

phosphate (0.0→10.0 mM) in 0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K (λexc = 395 nm).

(B) Changes in the integrated Eu(III) emission as a function of disodium phosphate, measured

between 570 – 720 nm.

Figure S33. Phosphorescence studies showing the effect of PO43− on AuNP-1.Eu.Na after 12 hr.

(A) Phosphorescent response of AuNP-1.Eu.Na (1 × 10−7 M) and Na2HPO4 (10 mM) as a

function of time (0→12 hr) in 0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K (λexc = 395 nm).


720 nm.

0.0 2.0x10-3

4.0x10-3

6.0x10-3

8.0x10-3

1.0x10-2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

I/I 0

Conc. of Na2HPO4 (M)

720580 600 620 640 660 680 700

0

10

20

30

40

50

60

70

800 M Na2HPO4

1 × 10-3 M Na2HPO4

1 × 10-2 M Na2HPO4

Inte

nsi

ty(a

.u)

Wavelength (nm)

580 600 620 640 660 680 700 720

0

10

20

30

40

50

60

70

800 M Na2HPO4

1 × 10-3 M Na2HPO4

1 × 10-2 M Na2HPO4 (+12 h)

Inte

nsi

ty(a

.u.)

Wavelength (nm)

0 1 2 3 4 5 6 7 8 9 10 11 12

0.0

0.2

0.4

0.6

0.8

1.0

1.2

I/I 0

Time (hr)

A B

A B

Figure S34. Formation of a 1:1 ternary complex in solution, 1.Eu-nta.

(A) Phosphorescent response of 1.Eu (1 × 10−5 M) upon titrating with nta (0.0→4.0 eq.) in

CH3OH (TBAP = 0.1 M) at 298 K (λexc = 330 nm).


617 nm.

Figure S35. Absorption studies showing the formation of 1:1 ternary complexes in solution on


(A) Changes in the absorbance of AuNP-1.Eu (1 × 10−7 M) upon titrating with nta (0→500 eq.)

in CH3OH (TBAP = 0.1) at 298 K. Inset: Magnified view.

(B) Changes in the SPR band of AuNP-1.Eu as a function of nta equivalents, measured at

522 nm.

580 600 620 640 660 680 700 720

0

50

100

150

200

250

300

350

Inte

nsi

ty (

a.u

.)

Wavelength (nm)

0.0 eq. nta

1.0 eq. nta

4.0 eq. nta

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

0

50

100

150

200

250

300

350

Inte

nsi

ty (

a.u

.)

Eq. of nta added

1 ± 0.1 eq. of nta

400 450 500 550 600 650 7000.000

0.008

0.016

0.024

0.032

Ab

sorb

an

ce

Wavelength (nm)

0 eq. nta

100 eq. nta

500 eq. nta

250 300 350 400 450 500 550 600 650 700

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Ab

sorb

an

ce

Wavelength (nm)

0 50 100 150 200 250 300 350 400 450 500

0.000

0.004

0.008

0.012

0.016

0.020

0.024

0.028

Ab

sorb

an

ce

Eq. of nta added

A B

A B

Figure S36. Phosphorescence studies showing the formation of 1:1 ternary complexes in

solution on the surface of AuNPs.

(A) The phosphorescent response of AuNP-1.Eu (1 × 10−7 M) upon titrating with nta (0→500 eq.)

in CH3OH (TBAP = 0.1 M) at 298 K (λexc = 330 nm).


617 nm.

Figure S37. Fluorescence studies showing the formation of 1:1 ternary complexes in solution on


(A) Fluorescent response of AuNP-1.Eu (1 × 10−7 M) upon titrating with nta (0→500 eq.) in

CH3OH (TBAP = 0.1 M) at 298 K (λexc = 330 nm).

(B) Changes in the antenna and Eu(III) emission as a function of nta equivalents, measured at

480 and 616 nm.

580 600 620 640 660 680 700 720

0

30

60

90

120

150

180

210

Inte

nsi

ty (

a.u

.)

Wavelength (nm)

0 eq. nta

100 eq. nta

500 eq. nta

0 50 100 150 200 250 300 350 400 450 500

0

20

40

60

80

100

120

140

160

180

200

100 10 eq. of nta

Inte

nsi

ty (

a.u

.)

Eq. of nta added

400 450 500 550 600 650 700

0

100

200

300

400

500

600

700

Inte

nsi

ty(a

.u.)

Wavelength (nm)

0 eq. nta

100 eq. nta

500 eq. nta

0 50 100 150 200 250 300 350 400 450 500

0

100

200

300

400

500

600

700

617 nm

480 nm

Inte

nsi

ty(a

.u.)

Eq. of nta added

A B

A B

Figure S38. Absorption studies showing the effect of PO43− on AuNP-1.Eu.

(A) Changes in the absorbance of AuNP-1.Eu (1 × 10−7 M) upon titrating with tetrabutyl-

ammonium phosphate (0.0→10.0 mM) in CH3OH at 298 K (λexc = 395 nm).

(B) Changes in the SPR band as a function of tetrabutylammonium phosphate, measured at

525 nm.

Figure S39. Phosphorescence studies showing the effect of PO43− on AuNP-1.Eu.

(A) Phosphorescent response of AuNP-1.Eu (1 × 10−7 M) upon titrating with tetrabutyl-

ammonium phosphate (0.0→0.0 mM) in 0.1 M HEPES (I = 0.1) at pH 7.4 and 298 K (λexc =

395 nm).

(B) Changes in the integrated Eu(III) emission as a function of tetrabutylammonium phosphate,

measured between 570 – 720 nm.

250 300 350 400 450 500 550 600 650 700

0.00

0.01

0.02

0.03

0.04

0.05

0.060 M TBAP

1 × 10−3 M TBAP

1 × 10−2 M TBAP

Ab

sorb

an

ce

Wavelength (nm)

0.0 2.0x10-3

4.0x10-3

6.0x10-3

8.0x10-3

1.0x10-2

0.015

0.020

0.025

0.030

0.035

0.040

Ab

sorb

an

ceConc. of TBAP (M)

0.0 2.0x10-3

4.0x10-3

6.0x10-3

8.0x10-3

1.0x10-2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

I/I 0

Conc. of TBAP (M)

580 600 620 640 660 680 700 720

0

30

60

90

120

150

1800 M TBAP

Inte

nsi

ty (

a.u

.)

Wavelength (nm)

1 × 10−3 M TBAP

1 × 10−2 M TBAP

A B

A B

Figure S40. Phosphorescence studies showing the effect of PO43− on AuNP-1.Eu after 12 hr.

(A) Phosphorescent response of AuNP-1.Eu (1 × 10−7 M) and tetrabutylammonium phosphate

(10 mM) as a function of time (0→12 hr) in CH3OH at 298 K (λexc = 395 nm).


720 nm.

2. Solid-State Photophysical Measurements

Figure S41. Phosphorescence studies of microdamaged bovine bone structure as a function of

time immersed in AuNP-1.Eu.Na.

(A) Concentration of AuNP-1.Eu.Na in 0.1 M HEPES (I = 0.1) at pH 7.4 = 3.1 × 10−4 M; λexc =

395 nm.

(B) Concentration of AuNP-1.Eu.Na in 0.1 M HEPES (I = 0.1) at pH 7.4 = 1.6 × 10−4 M; λexc =

395 nm.

Comparison between the surface of healthy bone being immersed and not immersed in

AuNP-1.Eu.Na is also given.

580 600 620 640 660 680 700 720

0

20

40

60

80

100

120

140 0 M TBAP

1.0 × 10–3 M TBAP

1.0 × 10–2 M TBAP

Inte

nsi

ty (

a.u

.)

Wavelength (nm)

0 1 2 3 4 5 6 7 8 9 10 11 12

0.0

0.2

0.4

0.6

0.8

1.0

1.2

I/I 0

Time (hr)

580 600 620 640 660 680 700 720

0

7000000

14000000

21000000

28000000

35000000

42000000

Inte

nsi

ty(c

ps)

Wavelength (nm)

24 hr

4 hr

0 hr

Surface immersed

Surface not immersed

580 600 620 640 660 680 700 720

0

10000000

20000000

30000000

40000000

50000000

Inte

nsi

ty(c

ps)

Wavelength (nm)

24 hr

4 hr

0 hr

Surface immersed


A B

A B

Figure S42. Luminescence studies of microdamaged bovine bone structure after immersion in

aqueous solution of AuNP-1.Eu.Na and nta.

(A) Phosphorescent response (λexc = 330 nm) after immersing the same scratched bone

specimen (already immersed in AuNP-1.Eu.Na) in nta (1.5 × 10−4 M).

(B) Fluorescent response (λexc = 330 nm) after immersing the same scratched bone specimen

(already immersed in AuNP-1.Eu.Na) in nta (1.5 × 10−4 M).

Figure S43. Phosphorescence studies of microdamaged bovine bone structure as a function of

time immersed in AuNP-1.Eu.

(A) Concentration of AuNP-1.Eu in CH3OH = 3.1 × 10−4 M; λexc = 395 nm.

(B) Concentration of AuNP-1.Eu in CH3OH = 1.6 × 10−4 M; λexc = 395 nm.

Comparison between the surface of healthy bone being immersed and not immersed in

AuNP-1.Eu is also given.

580 600 620 640 660 680 700 720

0

30000000

60000000

90000000

120000000

150000000

180000000

Inte

nsi

ty(c

ps)

Wavelength (nm)

24 hr

4 hr

0 hr

Surface immersed


400 450 500 550 600 650 700

0

600000

1200000

1800000

2400000

3000000

3600000

Inte

nsi

ty(c

ps)

Wavelength (nm)

24 hr

4 hr

0 hr

Surface immersed


580 600 620 640 660 680 700 720

0

5000000

10000000

15000000

20000000

25000000

Inte

nsi

ty(c

ps)

Wavelength (nm)

24 hr

4 hr

0 hr

Surface immersed


580 600 620 640 660 680 700 720

0

5000000

10000000

15000000

20000000

25000000

30000000

Inte

nsi

ty(c

ps)

Wavelength (nm)

24 hr

4 hr

0 hr

Surface immersed


A B

A B

Figure S44. Luminescence studies of microdamaged bovine bone structure after immersion in

a CH3OH solution of AuNP-1.Eu and nta.

(A) Phosphorescent response (λexc = 330 nm) after immersing the same scratched bone

specimen (already immersed in AuNP-1.Eu) in nta (1.5 × 10−4 M).

(B) Fluorescent response (λexc = 330 nm) after immersing the same scratched bone specimen

(already immersed in AuNP-1.Eu) in nta (1.5 × 10−4 M).

400 450 500 550 600 650 700

0

1000000

2000000

3000000

4000000

5000000

Inte

nsi

ty(c

ps)

Wavelength (nm)

24 hr

4 hr

0 hr

Surface immersed


580 600 620 640 660 680 700 720

0

40000000

80000000

120000000

160000000

200000000

Inte

nsi

ty(c

ps)

Wavelength (nm)

24 hr

4 hr

0 hr

Surface immersed


A B

3. Microscopy Studies

Figure S45. Polarized light microscopy images of microdamaged bovine bone structure after

being immersed for 0, 4, and 24 hr in an aqueous solution of AuNP-1.Eu.Na.

(A) Concentration of AuNP-1.Eu.Na in 0.1 M HEPES (I = 0.1) at pH 7.4 = 1.6 × 10−4 M.

(B) Concentration of AuNP-1.Eu.Na in 0.1 M HEPES (I = 0.1) at pH 7.4 = 7.8 × 10−5 M.

All scale bars = 250 μm.

Figure S46. Epifluorescence microscopy images of microdamaged bovine bone structure after

being immersed for 0, 4, and 24 hr in an aqueous solution of AuNP-1.Eu.Na, followed by

immersion in an aqueous solution of nta (1.5 × 10−4 M) for 30 sec.

(A) Concentration of AuNP-1.Eu.Na in 0.1 M HEPES (I = 0.1) at pH 7.4 = 1.6 × 10−4 M.

(B) Concentration of AuNP-1.Eu.Na in 0.1 M HEPES (I = 0.1) at pH 7.4 = 7.8 × 10−5 M.

Each image was acquired using a LED source (λexc = 365 nm) and a 520 longpass emission

filter; all scale bars = 250 μm.

0 hr 4 hr 24 hr

0 hr 4 hr 24 hr

A

B

A

B

Figure S47. TPE fluorescence microscopy images of microdamaged bovine bone structure

after being immersed for 0, 4, and 24 hr in an aqueous solution of AuNP-1.Eu.Na (3.1 × 10−4 M),

followed by immersion in an aqueous solution of nta (1.5 × 10−4 M) for 30 sec.

(A) Images taken at the bottom of the microcrack.

(B) 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 = 750 nm) and a red channel emission

filter (λem = 565 – 610 nm); all scale bars = 150 μm; aqueous solution = buffered 0.1 M HEPES (I =

0.1 M NaCl, pH 7.4).

0 h

r4

hr

24

hr

A B C


after being immersed for 0, 4, and 24 hr in an aqueous solution of AuNP-1.Eu.Na (1.6 × 10−4 M),

followed by immersion in an aqueous solution of nta (1.5 × 10−4 M) for 30 sec.





filter (λem = 565 – 610 nm); all scale bars = 150 μm; aqueous solution = buffered 0.1 M HEPES (I =

0.1 M NaCl, pH 7.4).

0 h

r4

hr

24

hr

A B C

Figure S49. Polarized light microscopy images of microdamaged bovine bone structure after

being immersed for 0, 4, and 24 hr in a CH3OH solution of AuNP-1.Eu.

(A) Concentration of AuNP-1.Eu = 3.1 × 10−4 M.

(B) Concentration of AuNP-1.Eu = 1.6 × 10−4 M.

(C) Concentration of AuNP-1.Eu = 7.8 × 10−5 M.

All scale bars = 250 μm.

0 hr 4 hr 24 hr

A B C

Figure S50. Epifluorescence microscopy images of microdamaged bovine bone structure after

being immersed for 0, 4, and 24 hr in a CH3OH solution of AuNP-1.Eu, followed by immersion in

a CH3OH solution of nta (1.5 × 10−4 M) for 30 sec.



(C) Concentration of AuNP-1.Eu = 7.8 × 10−5 M.

Each image was acquired using a LED source (λexc = 365 nm) and a 520 longpass emission

filter; all scale bars = 250 μm.

0 hr 4 hr 24 hr

A B C


after being immersed for 0, 4, and 24 hr in a CH3OH solution of AuNP-1.Eu (3.1 × 10−4 M),

followed by immersion in a CH3OH solution of nta (1.5 × 10−4 M) for 30 sec.





filter (λem = 565 – 610 nm); all scale bars = 150 μm.

0 h

r4

hr

24

hr

A B C









0 h

r4

hr

24

hr

A B C

Figure S54. TPE microscopy 2D snapshot images of microdamaged bovine bone structure after





Figure S55. TPE microscopy 3D snapshot images of microdamaged bovine bone structure after





24

hr

0h

r4

hr

24

hr

0h

r4

hr

A B

A B

Figure S56. Phosphorescence spectra obtained from quantum yield determination.

(A) 1.Eu.Na (1.5 × 10−4 M).

(B) AuNP-1.Eu.Na (2.3 × 10−6 M).

Supplemental References

1. McMahon, B., Mauer, P., McCoy, C.P., Lee, T.C., and Gunnlaugsson, T. (2009). Selective

imaging of damaged bone structure (microcracks) using a targeting supramolecular Eu(III)

complex as a lanthanide luminescent contrast agent. J. Am. Chem. Soc. 131, 17542–17543.

2. McMahon, B. K., and Gunnlaugsson, T. (2010). Lanthanide luminescence sensing of copper

and mercury ions using an iminodiacetate-based Tb(III)-cyclen chemosensor. Tetrahedron

Lett. 51, 5406.

3. Comby, S., and Gunnlaugsson, T. (2011). Luminescent lanthanide-functionalized gold

nanoparticles: exploiting the interaction with bovine serum albumin for potential sensing

applications. ACS. Nano. 5, 7184–7197.

4. Truman, L.K., Comby, S., and Gunnlaugsson, T. (2012). pH-responsive luminescent

lanthanide-functionalized gold nanoparticles with "on-off" ytterbium switchable near-

infrared emission. Angew. Chem. Int. Ed. 51, 9624–9627.

5. Massue, J., Quinn, S.J., and Gunnlaugsson, T. (2008). Lanthanide luminescent displacement

assays: the sensing of phosphate anions using Eu(III)-cyclen-conjugated gold nanoparticles

in aqueous solution. J. Am. Chem. Soc. 130, 6900–6901.

6. Yokokawa, S., Tamada, K., Ito, E., and Hara, M. (2003). Cationic self-assembled monolayers

composed of gemini-structured dithiol on gold: A new concept for molecular recognition

because of the distance between adsorption sites. J. Phys. Chem. B. 107, 3544–3551.

7. Massue, J., Plush, S.E., Bonnet, C.S., Moore, D.A., and Gunnlaugsson, T. (2007). Selective

mono N-alkylations of cyclen in one step syntheses. Tett. Lett. 48, 8052–8055.

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