This journal is The Royal Society of Chemistry 214 Supporting Information for A Fluorescence Turn-On Sensor for the Detection of Palladium Ions that Operates Through In-Situ Generation of Palladium Nanoparticles DonghoKeum, Sungwoo Kim and Youngmi Kim* Department of Chemistry, Institute of Nanosensor and Biotechnology, Dankook University, 126 Jukjeon-dong, Yongin-si, Gyeonggi-do, 448-71, Korea youngmi@dankook.ac.kr Tel: +8231-85-3156 Fax: +8231-85-3148 Table of Contents: 1. Studies on Formation of PdNPs and TEM Images of PdNPs 2. Photophysical Properties of iodo-bodipy and H-BODIPY 3. Fluorescence Turn-on Response 4. Identification of Fluorescent Reaction Product 5. Control Experiments 6. Selectivity Studies 7. Kinetic Studies 8. Determination of Detection Limit 9. Fluorescence Turn-on Response of 1 with Various Palladium Species 1. Determination of Palladium Contents in Chemical Products 11. References S1
Absorbance This journal is The Royal Society of Chemistry 214 Experimentals General methods, materials, instrumentation and measurements Iodo-BODIPY 1 and H-BODIPY 2 were prepared according to literature procedures. 1 Aqueous solutions were freshly prepared with deionized water from a water purification system (Human Corp. Korea). All metal ions were used as chloride salts except for AgNO 3. UV-Vis absorption spectra were obtained on a Shimadzu UV-251 spectrophotometer. Fluorescence measurements were recorded on a Hitachi F-7 fluorescence spectrophotometer at 25 C using 1 mm quartz cuvettes with a path length of 1 cm. Fluorescence quantum yields were determined by standard methods, using rhodamine 6G (Φ F =.95 in EtOH) 2 for iodo-bodipy 1 and fluorescein (Φ F =.95 in.1 M NaOH) 3 for H-BODIPY 2 as standards, respectively. Fluorometric assays with various metal analytes were measured by monitoring changes in fluorescence intensity using a Synergy Mx Microplate Reader (BioTek, USA). 1. Studies on Formation of PdNPs and TEM Images of PdNPs (a) Time-dependent absorption spectra of Pd 2+ in (1:4, v/v) at 25 o C.36.27.18 min 5 min 1 min 15 min 3 min.9. 2 3 4 5 6 7 Wavelength / nm Figure S1. Time-dependent UV-Vis absorption spectra of PdCl 2 (5 µm) in (1:4, v/v) at 25 o C. S2
Absorbance This journal is The Royal Society of Chemistry 214 (b) Determination of conversion yield of Pd 2+ in (1:4, v/v) to PdNPs at 25 o C In order to determine the correlation of absorbance and concentration of PdCl 2, UV-Vis absorption spectra of PdCl 2 at various concentrations in water was measured. The extinction coefficient of absorbance at 28 nm and concentration of PdCl 2 was calculated from the slope of a plot of the absorbance at 28 nm versus concentration of PdCl 2 in water. The calculated coefficient was used in determining the conversion yield of PdCl 2 in (1:4, v/v) to PdNPs. For example, the conversion yield was determined by measuring absorbance at 28 nm of supernatant after centrifugation of the suspended PdNPs that are readily generated by mixing PdCl 2 in aqueous ethanol, and comparing it with an absorbance vs. concentration calibration curve ( abs = 28 nm) for PdCl 2 in water. PdCl 2 (5 M) was converted to PdNPs in aqueous ethanol in ca. 9% yield..84.63.42.21 [PdCl2] in water Supernatant. 2 3 4 5 6 7 Wavelength / nm.6.4.2. 2 4 6 8 1 [PdCl 2 ] / M Absorbance at 28 nm.8 Equation Weight y = a + b*x No Weightin Residual.139 Sum of Squares Adj. R-Squar.99765 Value Standard Err B Intercept -.271.71 B Slope.772 1.18445E-4 Figure S2. (A) UV-Vis absorption spectra of PdCl 2 (1-1 M) in water at 25 o C. (B) A plot of absorbance at 28 nm vs [PdCl 2 ] in water at 25 o C. UV-Vis absorption spectra (red line) of the supernatant after centrifugation of the suspended PdNPs that were obtained 3 min after mixing PdCl 2 (5 M) in aqueous ethanol, were shown in Figure S2(A). S3
Frequency / % This journal is The Royal Society of Chemistry 214 (c) Transmission electron microscopy (TEM) analysis (A) (B) 4 3 2 1 2 3 4 5 6 7 Particle diameter / nm Figure S3. TEM image (A) and size distribution (B) of in-situ generated PdNPs from a solution of PdCl 2 (5 μm) in (1:4, v/v) at 25 o C. Average diameter of PdNPs is 4.2.2 nm. 2. Photophysical Properties of iodo-bodipy and H-BODIPY Table S1. Photophysical properties of iodo-bodipy 1 and H-BODIPY 2 a Compounds abs. max, nm, M -1 cm -1 em. max, nm b c Φ FL iodo-bodipy 1 535 51964 555.17 H-BODIPY 2 51 9298 512.55 a Data were obtained in (1:4, v/v) at 25 o C. b Excited at 465 nm c Quantum yields vs. rhodamine 6G in EtOH (Φ F =.95) for iodo-bodipy 1 2 and fluorescein in.1 N NaOH (Φ F =.95) for H-BODIPY 2. 3 S4
Fluorescence Intensity / a.u. Absorbance Fluorescence Intensity / a.u. This journal is The Royal Society of Chemistry 214 3. Fluorescence Turn-on Response Preparation of stock solution and general procedure: PdCl 2 dissolved in deionized water (.5 mm, 2 µl) was mixed with a mixture solution of ethanol and water (1:7, v/v, 16 µl). To the solution was added iodo- BODIPY 1 (.5 mm in EtOH, 2 µl) to have final concentration of 5 µm iodo-bodipy 1 and 5 µm PdCl 2 in ethanol and water (1:4, v/v), respectively. The reactions were monitored at 25 o C for 3 minutes. The fluorescence signal for each well was measured at 51 nm (λ ex = 465 nm). (a) Fluorescence turn-on response of 1 in the presence of PdCl 2 in.24.18 Without PdCl2 With PdCl2 6 45 Without PdCl2 With PdCl2.12 3.6 15. 3 4 5 6 7 8 Wavelength / nm 5 55 6 65 7 Wavelength / nm Figure S4. (left) Absortion and (right) fluorescence emission spectra of iodo-bodipy 1 (5 μm) without (black) and with (red) addition of Pd 2+ (5 μm) in (1:4, v/v) at 25 o C. The spectra were acquired 3 min after the addition of 1 to the solution of Pd 2+ in (1:4, v/v). Excited at 465 nm. (b) Time-dependent fluorescence turn-on response of 1 in the presence of different concentrations of Pd 2+ in 15 12 9 6 3 1 M [Pd 2+ ] F/F 5 4 3 2 1 5 1 15 2 25 3 2 4 6 8 1 [Pd 2+ ] / M Figure S5. (left) Time-dependent fluorescence intensity at 51 nm of 1 (5 µm) in the presence of different concentrations of Pd 2+ in ethanol water (1:4, v/v) at 25 o C. [Pd 2+ ] =,.5, 1, 5, 1, 15, 2, 25, 3, 35, 4, 45, 5, 6, 7, 8, 9, 1 M. Excited at 465 nm. The values were obtained every 2 min ( 3 min). (right) A plot of the relative fluorescence intensity at 51 nm as a function of [Pd 2+ ]. Incubation time = 3 min. F and F correspond to the fluorescence intensity of iodo-bodipy 1 in the absence and the presence of PdCl 2 in (1:4, v/v), respectively. S5
Abundance / % Abundance / % This journal is The Royal Society of Chemistry 214 4. Identification of Fluorescent Reaction Product Figure S6. HPLC chromatograms of 1 before (top); after addition of Pd 2+ (5 M) in ethanol water (1:4, v/v) at 25 o C (middle); 2 only (bottom).the samples were analyzed by using HPLC-MS with a linear gradient elution (eluent A/B =2/8, A: deionized water, B: Methanol with 5 mm ammonium formate, flow rate.3 ml/min, UV: 5 nm). [1] = [2] = 5 M, [Pd 2+ ] = 5 M. 1 8 (a) 918.2 [1 + NH 4 ] + 1 8 (b) 6 6 4 2 4 2 666.4 [2 + NH 4 ] + 4 5 6 7 8 9 1 m/z 4 5 6 7 8 9 1 m/z Figure S7. ESI-MS spectra of the peak of retention time at 12. min (a) and 4.5 min (b). The MW of the material eluting with a retention time of 12. min is 918.2, which corresponds to [M+NH 4 ] + for iodo- BODIPY 1 and the MW of the substance with a retention time of 4.5 min is 666.4, which corresponds to [M+NH 4 ] + for 2. S6
Fluorescence Intensity / a.u. Fluorescence Intensity / a.u. Fluorescence Intensity / a.u. This journal is The Royal Society of Chemistry 214 5. Control Experiments (a) Fluorescence turn-on response of 1 in the presence of PdCl 2 in various aqueous media 1 6 75 45 Ethanol, 1-Propanol, Isopropyl alcohol 5 25 Acetone DMSO Acetonitrile 3-Amino-1-propanol Ethanol 1-Propanol Isopropyl alcohol 5 1 15 2 25 3 3 15 Acetone, Acetonitrile, DMSO 3-Amino-1-propanol 5 55 6 65 7 Wavelength / nm Figure S8. (left) Time-dependent fluorescence intensity at 51 nm of 1 upon addition of Pd 2+ in water containing different organic solvents (2%) as a cosolvent at 25 o C. (right) Fluorescence emission spectra of iodo-bodipy 1 upon addition of Pd 2+ in water containing different organic solvents (2%) as a cosolvent at 25 o C. The spectra were acquired 3 min after the addition of 1 to the solution of Pd 2+ in aqueous media. Excited at 465 nm. [1] = 5 µm, [PdCl 2 ] = 5 μm (b) Effect of reducing agent (NaBH 4 ) on the iodo-bodipy 1 only 1 1 + NaBH 4 (.5 mm) 5 55 6 65 7 Wavelength (nm) Figure S9. Fluorescence emission spectra of iodo-bodipy 1 upon addition of NaBH 4 (5 M) in ethanol water (1:4, v/v) at 25 o C. Excited at 465 nm. S7
Fluorescence Intensity / a.u. Fluorescence Intensity / a.u. This journal is The Royal Society of Chemistry 214 (c) Stability of deiodinated product, H-BODIPY 2 in the presence of Pd 2+ 4 3 only 2 2 + Pd 2+ in 2 1 5 55 6 65 7 Wavelength / nm Figure S1. Fluorescence emission spectra of H-BODIPY 2 (5 μm) upon addition of Pd 2+ (5 μm) in ethanol water (1:4, v/v) at 25 o C. The spectra were obtained 3 min after the addition of Pd 2+ to the solution of H-BODIPY 2. Excited at 465 nm. (d) Photostability of iodo-bodipy 1 We have investigated photostability of 1 in ethanol water (1:4, v/v) at 25 o C.The photooxidation studies were performed by continuous visible light irradiation using a 15 W steady-state Xe lamp as the irradiation source under aerobic conditions. The photostability was quantified by monitoring the change of fluorescence intensity of 1 after photoirradiation for 2.5 hours. 16 12 min 15 min Irradiation 8 4 5 55 6 65 7 Wavelength / nm Figure S11. Fluorescence emission spectra of iodo-bodipy 1 (5 M) in ethanol water (1:4, v/v) at 25 o C before and after photoirradiation ( ex =465 nm) for 2.5 h. S8
This journal is The Royal Society of Chemistry 214 6. Selectivity Studies 6 45 F/F 3 15 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 [Analyte] M Figure S12. Relative fluorescence intensities of iodo-bodipy 1 toward various metal ions (as their chloride salts except for AgNO 3 ) in ethanol water (1:4, v/v), measured 3 minutes after addition of each metal ion at 25 C. 1, iodo-bodipy 1 only; 2, Pd 2+ ; 3, Al 3+ ; 4, Ca 2+ ; 5, Cd 2+ ; 6, Cu 2+ ; 7, Co 2+ ; 8, Cr 2+ ; 9, Fe 2+ ; 1, Fe 3+ ; 11, Pb 2+ ; 12, Zn 2+ ; 13, Mg 2+ ; 14, Hg 2+ ; 15, Ni 2+ ; 16, Mn 2+ ; 17, K + ; 18, Ag + ; 19, Na + ; 2, Au + ; 21, Au 3+ ; 22, Pt 2+ [1] = 5 M, [metal ion] = 2 M for Pd 2+ and 5 M for all other metal ions. Excited at 465 nm. Figure S13. Photographs of probe 1 upon addition of various metal ions under (top) ambient light and (bottom) UV irradiation (365 nm). 1, iodo-bodipy 1 only; 2, Pd 2 +; 3, Al 3+ ; 4, Ca 2+ ; 5, Cd 2+ ; 6, Cu 2+ ; 7, Co 2+ ; 8, Cr 2+ ; 9, Fe 2+ ; 1, Fe 3+ ; 11, Pb 2+ ; 12, Zn 2+ ; 13, Mg 2+ ; 14, Hg 2+ ; 15, Ni 2+ ; 16, Mn 2+ ; 17, K + ; 18, Ag + ; 19, Na + ; 2, Au + ; 21, Au 3+ ; 22, Pt 2+ [1] = 5 M, [metal ion] = 2 M for Pd 2+ and 5 M for all other metal ions. S9
Fluorescence Intensity / a.u. This journal is The Royal Society of Chemistry 214 7. Kinetic Studies Standard fluorescence curve: In order to determine the correlation of fluorescence intensity and concentration of dehalogenation product 2, fluorescence intensities of various concentrations of 2 at 51 nm were measured. The extinction coefficient of fluorescence intensity and concentration of 2 was calculated from the slope of a plot of the fluorescence intensity versus concentration of 2. The calculated coefficient was used in determining kinetic constants of the conversion of iodo-bodipy 1 to H-BODIPY 2 by Pd 2+ in (1:4, v/v) at 25 o C. 25 2 Equation y = a + b*x.99676 Adj. R-Square Value Standard Error B Intercept 86.9524 276.41323 B Slope 7165.65714 182.59257 15 1 5..5 1. 1.5 2. 2.5 Figure S14. Standard fluorescence curve of H-BODIPY 2 in (1:4, v/v) at 25 o C. Fluorescence intensity at 51 nm was measured. Excited at 465 nm. Determination of kinetic constant: To determine kinetic constant, iodo-bodipy 1 (5 M) was added to the solution of Pd 2+ (5 M) in (1:4, v/v) at 25 o C. 1.5 Pd 2+ 5 M 1.5 Pd 2+ 5 M 1.125 1.125.75.375 1..75.5.25 Equation y = a + b* Weight No Weigh Residual.1157 Sum of Squares Adj. R-Sq.97812 Value Standard B Intercept.46.222 B Slope.144.684 k = 2894 M -1 min -1 Pd 2+ 5 M.75.375 1. Equatio y = a Adj. R-.972 Valu Standar.75 B Interc.4.2663 B Slope.17.9 k = 342 M -1 min -1.5.25 Pd 2+ 5 M. 1 2 3 4 5. 5 1 15 2 25 3. 1 2 3 4 5. 5 1 15 2 25 3 Figure S15. Kinetics for the fluorescence response of iodo-bodipy 1 (5 M) with Pd 2+ (5 M) in (1:4, v/v) at 25 o C. Fluorescence intensity was measured at 51 nm. Excited at 465 nm. S1
Equation y = a Adj. R-S.977 Value Standard B Interc -.1.418 B Slope.29.141 This journal is The Royal Society of Chemistry 214 (a) Several factors to improve kinetics: amount of ethanol in water 1..75.16.12.8 k = 595 M -1 min -1 (5:95) (5:95) 1..75 (1:9).4.5. 1 2 3 4 5.5.5.375 Equatio y = a Adj. R-S.998 Value Standard B Interc -.1.382 B Slope.9.129 (1:9).25.25 k = 1876 M -1 min -1.25.125. 5 1 15 2 25 3. 1 2 3 4 5. 5 1 15 2 25 3 1.5 1.125 (2:8) 1.5 1.125 (3:7).75.375 1.2.9.6.3 Equation y = a + Adj. R-Sq.8882 Value Standard B Interce.176.5188 B Slope.157.1754 k = 3146 M -1 min -1 (2:8).75.375 1.2 Equation y = a + Adj. R-Squ.9386 Value Standard E B Intercep.135.422 B Slope.168.136.9 k = 3373 M -1 min -1.6.3 (3:7). 1 2 3 4 5. 5 1 15 2 25 3. 1 2 3 4 5. 5 1 15 2 25 3 Figure S16. Kinetics for the fluorescence response of iodo-bodipy 1 (5 M) with Pd 2+ (5 M) in water containing different % ethanol (5-3%) as a cosolvent at 25 o C. Fluorescence intensity was measured at 51 nm. Excited at 465 nm. (b) Several factors to improve kinetics: temperature 1.3 at 5 o C.975.65 1.5 1.125 Equatio y = a Adj. R-S.967 Valu Standard B Interc.5.3488 B Slope.2.1179 at 5 o C.325.75.375 k = 447 M -1 min -1. 1 2 3 4 5. 5 1 15 2 25 3 Figure S17. Kinetics for the fluorescence response of iodo-bodipy 1 (5 M) with Pd 2+ (5 M) in (1:4, v/v) at 5 o C. Fluorescence intensity was measured at 51 nm. Excited at 465 nm. S11
(c) Several factors to improve kinetics: additional reducing agent, NaBH 4 Fluorescence Intensity / a.u. This journal is The Royal Society of Chemistry 214 4 3 2 With NaBH 4 Without NaBH 4 1 5 1 15 2 25 3 Figure S18. Time-dependent fluorescence intensity at 51 nm of 1 (5 µm) upon addition of Pd 2+ (5 µm) in the presence of NaBH 4 (.5 mm) in ethanol water (1:4, v/v) at 25 o C. Excited at 465 nm. 8. Determination of Detection Limit 12 R 2 =.989 9 F/F 6 3..2.4.6.8.1 [Pd 2+ ] / M Figure S19. A plot of relative fluorescence intensity at 51 nm of iodo-bodipy 1 (5 M) as a function of [Pd 2+ ]. Fluorescence spectra of iodo-bodipy 1 (5 M) were measured 3 min after addition of Pd 2+ in ethanol water (1:4, v/v,.5 mm NaBH 4 ) at 25 o C. Excited at 465 nm. F and F correspond to the fluorescence intensity of iodo-bodipy 1 in the absence and the presence of PdCl 2 in (1:4, v/v,.5 mm NaBH 4 ), respectively. [Pd 2+ ] = -.1 M S12
This journal is The Royal Society of Chemistry 214 9. Fluorescence Turn-on Response of 1 with Various Palladium Species 13 975 F/F 65 325 1 2 3 4 5 6 7 [Palladium species] / M Figure S2. Relative fluorescence intensities at 51 nm of iodo-bodipy 1 (5 M) toward various different palladium species (5 M) in ethanol H 2 O (1:4, v/v,.5 mm NaBH 4 ), measured 3 minutes after addition of each analyte at 25 C. 1, Pd(NO 3 ) 2 ; 2, Pd(OAc) 2 ; 3, PdCl 2 ; 4, Na 2 PdCl 4 ; 5, Pd(PPh 3 ) 4 ; 6, PdCl 2 (PPh 3 ) 2 ; 7, PdCl 2 (dppf) 2. Excited at 465 nm. S13
This journal is The Royal Society of Chemistry 214 1. Determination of Palladium Contents in Chemical Products Biphenyl derivative 3 was synthesized by a Suzuki-Miyaura cross coupling reaction of an arylboronate (21 mg, 1.1 mmol) and an aryl halide (3 mg, 1.36 mmol) in the presence of Pd(PPh 3 ) 4 (81 mg,.7 mmol) and purified by using column chromatography (Scheme S1). Standard curve was obtained by measuring fluorescence intensity at 51 nm of iodo-bodipy 1 (5 M) 3 min after the addition of Pd(PPh 3 ) 4 at various concentrations (1-5 M) in ethanol H 2 O (1:4, v/v,.5 mm NaBH 4 ) at 25 o C. For the determination of amount of Pd in the synthesized 3, biphenyl derivative 3 (1.9 mg) was dissolved in ethanol (1 ml). An aliquot of this solution (2 µl) was mixed with a solution of NaBH 4 in water (16 µl, final [NaBH 4 ] =.5 mm). To the solution was added a solution of iodo-bodipy 1 in EtOH (2µL, [1] =.5 mm) to result in an assay solution nominally 5 µm in 1. The reactions were monitored at 25 o C for 3 minutes in a microplate reader, during which the fluorescence signal for each well was measured at 51 nm (λ ex = 465 nm). Comparison of the fluorescence signals from analysis of 3 (38 g), obtained using sensing system comprised of iodo-bodipy 1 (5 M) in ethanol H 2 O (1:4, v/v,.5 mm NaBH 4,) at 25 o C, with those from a standard curve show that that the purified biphenyl derivative 3 (38 g) contains 2.1 ppm of Pd. Scheme S1. Synthesis of compound 3. Figure S21. Determination of residual palladium contents in biphenyl derivative 3 (38 g). Comparison of the fluorescence signals from analysis of 3 (red circle), obtained using sensing system comprised of iodo- BODIPY 1 (5 M) in ethanol H 2 O (1:4, v/v,.5 mm NaBH 4, 25 o C), with those from a standard curve (black circle). Excited at 465 nm. The measured [Pd] in 3 was determined from the average of 4 independent measurements to be 2.1 ppm 11. References 1 J. Park, S. Choi, T.-I. Kim and Y. Kim, Analyst, 212, 137, 4411. 2 R. F. Kubin and A. N. Fletcher, J. Luminescence, 1982, 27, 455. 3 A. T. R. Williams, S. A. Winfield and J. N. Miller, Analyst, 1983, 18, 167. S14