Supporting Information

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1 Supporting Information A Facile and Sensitive Near-Infrared Fluorescence Probe for the Detection of Endogenous Alkaline Phosphatase Activity in Vivo Song-Jiao Li, Chun-Yan Li,*,, Yong-Fei Li, Junjie Fei, Ping Wu, Bin Yang, Juan Ou-Yang, and Shi-Xin Nie Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, , People s Republic of China College of Chemical Engineering, Xiangtan University, Xiangtan, , People s Republic of China State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha, , People s Republic of China *Corresponding Author. lichunyan79@sina.com. Fax: S-1

2 Table of contents 1. Characterization of CyP S-3 2. Effects of ph and temperature S-5 3. Effect of incubation time S-6 4. Enzyme kinetics S-7 5. Selectivity S-8 6. ALP inhibitor investigation S-9 7. Effect of inhibitor on CyOH S HPLC experiments S Mass spectra of CyP reacted with ALP S DFT calculations S Cell viability S Comparison of ALP probes S-15 S-2

3 1. Characterization of CyP Figure S1. 1 H NMR spectra of CyP in CDCl3. Figure S2. 13 C NMR spectra of CyP in CDCl3. S-3

4 Figure S3. 31 P NMR spectra of CyP in CDCl3. Figure S4. Mass spectra of CyP. S-4

5 2. Effects of ph and temperature Figure S5. Effect of ph on the fluorescence of CyP (10 μm) before and after reaction with ALP (2.0 U/mL). λex / λem = 690 / 738 nm. Figure S6. Effect of temperature on the fluorescence of CyP (10 μm) before and after reaction with ALP (2.0 U/mL). λex / λem = 690 / 738 nm. S-5

6 3. Effect of incubation time Figure S7. Fluorescence spectra of CyP (10 µm) upon the addition of ALP (2.0 U/mL) in Tris-HCl buffer solution (50 mm, ph 8.0) at 37 C recorded every 2 min. λex = 690 nm. Inset: the plot of fluorescence intensity versus time. S-6

7 4. Enzyme kinetics Figure S8. (a) Michaelis-Menten plot and (b) Lineweaver-Burke plot for the reaction between CyP and ALP (2.0 U/mL) in Tris-HCl buffer solution (50 mm, ph 8.0) at 37 C. λex / λem = 690 / 738 nm. S-7

8 5. Selectivity Figure S9. Fluorescence response of CyP (10 µm) to ALP (2 U/mL), biothiols (1 mm), amino acids (1 mm) and metal ions (1 mm) in Tris-HCl buffer (50 mm, ph 8.0) at 37 C recorded within 20 min. 1. Blank, 2. ALP, 3. GSH, 4. Cys, 5. Hcy, 6. Pro, 7. Tyr, 8. Asn, 9. Met, 10. Phe, 11. Lue, 12. Asp, 13. Orn, 14. Ala, 15. Trp, 16. His, 17. Ser, 18. Gly, 19. Val, 20. Lys, 21. Glu, 22. Arg, 23. Ileu, 24. Thr, 25. K +, 26. Ca 2+, 27. Zn 2+, 28. Na +, 29. Mg 2+. λex / λem = 690 / 738 nm. S-8

9 6. ALP inhibitor investigation Figure S10. Fluorescence spectra of CyP (10 μm) in the presence of ALP (2.0 U/mL) at different Na3VO4 concentrations (0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 µm) in Tris-HCl buffer solution (50 mm, ph 8.0) at 37 C recorded at 20 min. λex = 690 nm. S-9

10 7. Effect of inhibitor on CyOH Figure S11. Effect of inhibitor (Na3VO4) on the fluorescence of CyOH. λex = 690 nm. S-10

11 8. HPLC experiments Figure S12. HPLC chromatograms of (a) CyOH, (b) CyP and (c) CyP reacted with ALP. The HPLC mobile phase was as follows: solvent A (CH3CN), solvent B (0.025 M ammonium acetate buffer), A/B=17/3 (v/v). S-11

12 9. Mass spectra of CyP reacted with ALP Figure S13. Mass spectra of CyP reacted with ALP. S-12

13 10. DFT calculations Figure S14. The optimized structures of CyP and CyOH. In the ball-and-stick model, carbon, oxygen and nitrogen atoms are colored in gray, red and blue, respectively. Figure S15. Frontier molecular orbitals of CyP and CyOH. S-13

14 11. Cell viability Figure S16. MTT assay for estimating cell viability (%) of HeLa cells treated with various concentrations of CyP (0-30 μm). S-14

15 12. Comparison of ALP probes Table S1. Comparison of this probe with other fluorescent probes for ALP Probe Chemical structure Ex/Em Detection limit Response time Application Ref /525 Not mentioned 30 min Cell imaging Cell and Ref / U/L 40 min zebrafish imaging Ref / U/L 300 s Serum Ref / U/L 15 min Cell imaging Ref / U/L 20 min Cell imaging Ref / U/mL 120 min Cell imaging This work 690/ U/mL 20 min Cell, tissue and living animal S-15