A novel terbium functionalized micelle nanoprobe for ratiometric fluorescence detection of anthrax spore biomarker

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1 Supporting Information for A novel terbium functionalized micelle nanoprobe for ratiometric fluorescence detection of anthrax spore biomarker Ke Luan, Ruiqian Meng, Changfu Shan, Jing Cao, Jianguo Jia, Weisheng Liu and Yu Tang* State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou , P. R. China * tangyu@lzu.edu.cn. Fax: S-1

2 Table of Contents 1. Experimental Methods A. Synthesis of FR. B. Preparation of the ligand L micelle C. Preparation of Tb 3+ -L micelle. D. General procedure for fluorescence measurement E. Calculating stability constant. F. Calculating coordinating water. 2. Supporting Figures and Tables Scheme S1. Synthesis of FR. Figure S1. Size distribution of (a) ligand L micelle, (b) Tb 3+ -L micelle, (c) Terbium functionalized micelle. Figure S2. TEM image of the terbium functionalized micelle. Figure S3. Absorption spectra (a) and FTIR spectra (b) of ligand L and Tb 3+ -L. Figure S4. TGA curve of terbium functionalized micelle. Figure S5. Effect of solution ph on the fluorescence (FL) intensity of FR. Figure S6. Absorption spectra of DPA,FL reference, terbium functionalized micelle and terbium functionalized micelle in the presence of DPA. Figure S7. Excitation and emission spectra of FR, terbium functionalized micelle and terbium functionalized micelle in the presence of DPA. Figure S8. Effect of solution ph on the FL intensity I 545 /I 440 of terbium functionalized micelle upon adding DPA. Figure S9. Linear plot of the fluorescence intensity of terbium functionalized micelle. Figure S10. (a) Titration profile according to the emission intensity of terbium functionalized micelle (b) Linear plot for calculating the stability constant. Figure S11. Luminescence decay curves of Tb 3+ -L in micelle. Figure S12. Luminescence decay curves of Tb 3+ -L in micelle in presence of DPA. Table S1. ζ potential of ligand L, Tb 3+ -L, terbium functionalized micelle and terbium S-2

3 functionalized micelle upon adding 100 µm DPA. Table S2. Merits, detection limits and linear range of lanthanide(iii)-based probes for measuring DPA. 3. NMR spectra and ESI-MS of the synthesized ligand and FR 4. Supporting References S-3

4 1. Experimental Methods A. Synthesis of FR. Scheme S1. Synthesis of FR The synthesis routs of FR as shown in Scheme S2. Compound FR was synthesized according to the literature methods. S1 B. Preparation of the ligand L micelle Ligand L (28.6 mg, 0.05 mmol) was dissolved in water (2 ml) and stirred overnight at room temperature. Then the reaction mixture was dialyzed with a cellulose ester dialysis membrane (500 MWCO) for 2 days to completely remove ligand L. The resulting material was dried by lyophilization to obtain micelle powder. C. Preparation of Tb 3+ -L micelle Ligand L (28.6 mg, 0.05 mmol) was dissolved in water (2 ml) and stirred at room temperature. A solution of terbium (III) nitrate hexahydrate (22.5 mg, 0.05 mmol) in water (2 ml) was added dropwise to the above solution. The reaction mixture was stirred overnight at room temperature. Then the reaction mixture was dialyzed with a cellulose ester dialysis membrane (500 MWCO) for 2 days to completely remove free Tb 3+ ions and ligand L. The resulting material was dried by lyophilization to obtain micelle powder, which was dispersed in water (1 mg ml -1 ) for further characterization and use. D. General procedure for fluorescence measurement The fluorescence spectra were recorded in the range of nm with excitation at 275 S-4

5 nm. 100 µl of stock solution of micelle (1 mg ml -1 ) was dispersed with 1.9 ml of HEPES buffer solution (10 mm, ph = 7.0), then various concentration of DPA (0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 15.0, 18.0 µm; aqueous solution) were added keeping constant the total volume of the mixture, and the fluorescence intensity of each solution was measured. E. Calculating stability constant According to the change of intensity located at the maximum emission wavelength versus the concentration of DPA, the stability constant of micelles with DPA can be calculated by using the equation as follow. (1) where, F 0 and F represent the fluorescent intensity of the micelles in the absence or presence of DPA while F max represents the maximum value of fluorescent intensity of micelles in the presence of DPA. [DPA] is the concentration of added DPA. n is the number of bound DPA molecules per micelles. K S is the stability constant of micelles with DPA. F. Calculating coordinating water. As previously mentioned, the fluorescence of Tb 3+ ions in micelle was quenched due to the non-radiative transition of the excited state of Tb 3+ ions to the hydroxyl stretching vibration of coordinating water molecules. In this case, when the coordinating water molecules are replaced by D 2 O, the luminescence lifetime of Tb 3+ ions in the micelle will be increased. The number of water molecules coordinating to Tb 3+ ions, represented by q value, can be calculated by using formula as follows, S2 where and represent the rate constants S-5

6 of luminescence decay, which are measured in H 2 O and D 2 O, respectively (the decay curves are shown in Figures S13 and S14). =5.0 ( 0.06) (2) The luminescence lifetime of the Tb 3+ -L micelle in D 2 O (τ = ms) was longer than that in H 2 O (τ = ms), which confirmed the above-mentioned quenching mechanism. The value of q was calculated as 2.25, indicated that there are almost two water molecules coordinate with Tb 3+ ion in aqueous solution of micelle. Its fluorescence lifetime in water (τ = ms) and D 2 O (τ = ms) was much longer in the presence of DPA, and the characteristic fluorescence intensity of Tb 3+ ions was enhanced. The value of q was determined as 1.95 in the presence of DPA. S-6

7 2. Supporting Figures and Tables Figure S1. Size distribution of (a) ligand L micelle, (b) Tb 3+ -L micelle. Figure S2. (a) Absorption spectra of ligand L and ligand L + Tb(NO 3 ) 3, 10 mm HEPES buffer (10 mm, ph = 7.0). (b) FTIR spectra of ligand L and Tb 3+ -L. Table S1. ζ potential of ligand L, Tb 3+ -L, terbium functionalized micelle and terbium functionalized micelle upon adding 100 µm DPA in HEPES buffer (10 mm, ph = 7.0) Sample Ligand L Tb 3+ -L Micelle Micelle + DPA ζ potential (mv) S-7

8 Figure S3. TGA curve of terbium functionalized micelle. Figure S4. Effect of solution ph on the fluorescence (FL) intensity of FR. Excitation wavelength: 275 nm; Emission wavelength: 440 nm. S-8

9 Figure S5. Absorption spectra of DPA (5 µm), FL reference, terbium functionalized micelle (50 µg ml -1 ) and micelle in the presence of 5 µm DPA. Figure S6. Excitation and emission spectra of FR, terbium functionalized micelle and terbium functionalized micelle in the presence of DPA, slit: 5 nm / 5 nm. S-9

10 Figure S7. TEM image of the terbium functionalized micelle upon addition of 100 µm DPA, the grid was poststained with uranyl acetate. Figure S8. Terbium functionalized micelle upon addition of 100 µm DPA in aqueous solution measured by DLS. S-10

11 Figure S9. Effect of solution ph on the FL intensity I 545 /I 440 of ratiometric micelle (50 µg ml -1 ) upon adding 4 µm DPA. Excitation wavelength: 275 nm. Figure S10. Linear plot of the fluorescence intensity of terbium functionalized micelle (50 µg ml -1 ) at 545 nm against DPA concentration (0-18 µm). S-11

12 Figure S11. (a) Titration profile according to the emission intensity of micelle at 545 nm in the presence of DPA. (b) Linear plot for calculating the stability constant. Figure S12. Luminescence decay curves of Tb 3+ -L in micelle. Yellow: decay curve obtained in H 2 O dispersion; Green: in D 2 O dispersion. λ ex = 275 nm, the decay curves were fitted with single exponential curves, χ 2 = 0.963, S-12

13 Figure S13. Luminescence decay curves of Tb 3+ -L in micelle in presence of DPA. Yellow: decay curve obtained in H 2 O dispersion; Green: in D 2 O dispersion. λ ex = 275 nm, the decay curves were fitted with single exponential curves, χ 2 = 0.979, Table S2. Luminescence Lifetime τ and the Rate Constant k Determined for [Tb 3+ -L] in H 2 O and D 2 O in the Presence and Absence of DPA, respectively Samples τ (ms) k (ms -1 ) q [Tb 3+ -L] (H 2 O) [Tb 3+ -L] (D 2 O) [Tb 3+ -L] (H 2 O) + DPA [Tb 3+ -L] (D 2 O) + DPA S-13

14 Table S3. Merits, detection limits and linear range of lanthanide(iii)-based probes for measuring DPA. Lanthanide(III)-based probe Single-walled carbon nanotube-tb Merits Optical and electrochemical Detection Linear range reference limit 100 nm 100 nm-1µm 5b FITC-doped silica nanoparticles-eu Ultrasensitive detection 0.2 nm 0.6 nm-600 nm 7b ratiometric 34nM - 12 Molecular printboards-eu glasss substrates 25 nm 25 nm-200 nm 13 Carbon dot-tb biocompatible 5 nm 5 nm-1.2 µm 11 Terbium functionalized micelle Ratiometric, biocompatible, broad linear range, rapid and sensitive detection 54 nm 0 µm-7 µm This work S-14

15 3. NMR spectra and ESI-MS of the synthesized ligand and FR 1 H NMR of 1-(4-dodecyloxy-phenyl)-ethanone in CDCl 3 1 H NMR of 3-(4-dodecyloxy-phenyl)-3-oxo-propionic acid methyl ester in CDCl 3 S-15

16 1 H NMR of N-(2-amino-ethyl)-3-(4-dodecyloxy-phenyl)-3-oxo-propionamide in CDCl 3 1 H NMR of the ligand L in DMSO-d 6 S-16

17 1 H NMR of FR in DMSO-d 6 13 C NMR of 1-(4-dodecyloxy-phenyl)-ethanone in CDCl 3 S-17

18 13 C NMR of 3-(4-dodecyloxy-phenyl)-3-oxo-propionic acid methyl ester in CDCl C NMR of N-(2-amino-ethyl)-3-(4-dodecyloxy-phenyl)-3-oxo-propionamide in CDCl 3 S-18

19 13 C NMR of the ligand L in DMSO-d 6 ESI-MS of 1-(4-dodecyloxy-phenyl)-ethanone S-19

20 ESI-MS of 3-(4-dodecyloxy-phenyl)-3-oxo-propionic acid methyl ester ESI-MS of N-(2-amino-ethyl)-3-(4-dodecyloxy-phenyl)-3-oxo-propionamide S-20

21 ESI-MS of the ligand L S-21

22 4. Supporting References (S1) Singha, S.; Kim, D.; Rao, A. S.; Wang, T.; Kim, K. H.; Lee, K.-H.; Kim, K.-T.; Ahn, K. H. Dyes Pigm. 2013, 99, (S2) Liu, J.; Morikawa, M.-a.; Kimizuka, N. J. Am. Chem. Soc. 2011, 133, S-22