Single Molecule Electrochemistry on a Porous Silica-Coated Electrode

Transcription

1 Supporting information for Single Molecule Electrochemistry on a Porous Silica-Coated Electrode Jin Lu, Yunshan Fan, Marco Howard, Joshua C. Vaughan, and Bo Zhang* Department of Chemistry, University of Washington, Seattle, Washington , United States Phone: Fax: zhang@chem.washington.edu Contents: Figure S1 Figure S2 Figure S3 Figure S4 Figure S5 Figure S6 Figure S7 Figure S8 Figure S9 Figure S10 Figure S11 Figure S12 References S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S13 S14 S14 S1

2 Figure S1. The fluorescent voltammetric responses of 500 nm resazurin (blue) and 500 nm resorufin (red) in 50 mm phosphate buffer (ph 7.4) and 1 M KCl on the bare ITO glass as the working electrode. The potential was cycled from 0 to -1 V (green) versus an Ag/AgCl reference electrode at the scan of 50 mv/s. CE: Pt wire. As the concentration of resazurin or resorufin was decreased to nanomolar, the redox current peaks of the processes are too small to be detected on the ITO electrode. Therefore, these electrochemical processes were only monitored by fluorescence, as shown in Fig. S1. A significant and reversible switch of fluorescent intensity is necessary to study the fluorogenic reactions at the single molecule level. When the weak-fluorescent resazurin was used as the starting reagent, the irreversible process only showed an obvious transition at the first potential cycle, and then completely disappeared at the 3 rd cycle (blue curve in Fig. S1). However, is a reversible electrochemical process, resulting in a more reversible switch of fluorescent intensity during the multiple potential cycles, especially when resorufin was used as the starting reagent (red curve in Fig. S1). Therefore, the fluorescent resorufin was selected, and its reversible reduction to non-fluorescent dihydroresorufin was studied in this report. S2

3 Figure S2. The fluorescence images of 10 nm ~ 10 pm resorufin solution on the bare ITO surface. Image size is 34 µm 34 µm. For a better comparison, the images were set to the same contrast value. The total fluorescence intensity decreased as the resorufin concentration decreased from 10 nm to 10 pm. However, no single molecule fluorescence was detected on the bare ITO surface. S3

4 Figure S3. The fluorescence images collected in H 2 O (a), 3.3 nm resorufin (b), and 3.3 nm dihydroresorufin (c) solutions on the mesoporous silica coated ITO surface. Dihydroresorufin was generated from a mixture of 3.3 nm resorufin, 33 µm glucose and 167 µm NaOH. 1 Image size is 27 µm 27 µm. For a better comparison, the images were set to the same contrast value. S4

5 Figure S4. Image of 1 nm resorufin on the mesoporous silica coated ITO surface before (a) and after (b) detecting all spots using ThunderSTORM. Image size is 20 µm 20 µm. S5

6 Figure S5. 3-D mesh surface (a) and fluorescence image (b) of single fluorescence burst. Image size: 9 pixel 9 pixel; pixel size: 160 nm. 2-D Gaussian fitting (c) and residual of the fitting (d) of the image in (b). S6

7 Figure S6. (a) Histogram of the lifetime of individual resorufin molecules before photobleaching. Solid line is the single-exponential decay fit with a time constant of 8.78 ± 2.97 s. (b) Histogram of blinking ontime of individual resorufin molecules. Solid line is the single-exponential decay fit with a time constant of 0.74 ± 0.03 s. To measure the photobleaching lifetime and the blinking on-time of individual resorufin molecules, we immobilized the resorufin molecules in a poly(methyl methacrylate) (PMMA) thin film by spin-coating 1 nm resorufin solution in 1 mg/ml PMMA in toluene on a piranha pretreated coverslip. The resorufin- PMMA coverslip was observed by a total internal reflection fluorescence microscope with 1.4 kw/cm 2 laser illumination (same laser power density used for single molecule fluorescence experiments). A video containing 3000 frames (10 frames per second) was recorded, and 311 resorufin molecules were detected on the first frame. Multiple blinking events were observed from the fluorescence trajectories of all the 311 single resorufin molecules, before the molecules were photobleached. The distributions of photobleaching lifetime and the blinking on-time of resorufin molecules were shown in Fig. S6, which were further fitted by a single-exponential decay function. S7

8 τ on (s) Laser power density (kw/cm 2 ) Figure S7. Laser power density dependence of τ on of 1 nm resorufin in 50 mm phosphate buffer (ph 7.4) and 1 M KCl solution on the mesoporous silica coated ITO surface. S8

9 # molecule cm Concentration (nm) Figure S8. The number of single molecular spots per cm 2 verses resorufin concentration ranging from 10 pm to 10 nm on the mesoporous silica coated ITO surface. The graph shows levelling off of the surface coverage ( ) as the resorufin concentration ([ ]) is increasing, which follows the Langmuir isotherm = [ ], where is the equilibrium constant. [ ] S9

10 Figure S9. The log-log plot of Fig. 5c in the main text. For a surface adsorption controlled electrochemical process, the slope of the linear fitting of ~ is close to 1. For a surface adsorption controlled reversible electrochemical process, the faradaic peak current ( ) of the curve is given by = Γ, where is the faraday constant, is the gas constant, is temperature, is the potential scan rate, is the electrode area, and Γ is the amounts of adsorbed per unit area. 2 The peak current ( ) is proportional to the scan rate ( ). Therefore, the slope of the linear fitting of ~ or ~ should be 1. S10

11 Figure S10. (a) The fluorescence intensity over three continuous potential sweeps with the scan rate of 50 mv/s, 100 mv/s, 200 mv/s, and 500 mv/s in 100 pm resorufin solution containing 50 mm phosphate buffer (ph 7.4) and 1 M KCl on the bare ITO electrode. (b) The time derivative of fluorescence intensity as a function of applied potential from 0 to -1 V at scan rate of 50 mv/s (orange), 100 mv/s (blue), 200 mv/s (magenta) and 500 mv/s (black). (c) The square root of scan rate dependence of the peak values (black square) of the time derivative traces in (b). Red lines are the linear fit. (d) The log-log plot of (c). The slope of the linear fitting of ~ is close to 0.5, indicating a diffusion controlled electrochemical process. As reported previously 3, the electrochemical current of a fluorogenic redox reaction is expressed by the change in fluorescence intensity, ~. For a diffusion controlled reversible electrochemical process, the faradaic peak current ( ) of the curve is given by = / / / /, where is the faraday constant, is the gas constant, is temperature, is the potential scan rate, is the electrode area, is the diffusion coefficient of species, and is the bulk concentration of species. 2 S11

12 The peak current ( ) is proportional to the square root of the scan rate ( / ). Therefore, the slope of the linear fitting of ~ or ~ should be 0.5. S12

13 Figure S11. (a) The number of single molecular spots detected on each frame over three continuous potential sweeps in 10 pm (orange), 100 pm (blue), 1 nm (magenta), and 10 nm (black) resorufin solution containing 50 mm phosphate buffer (ph 7.4) and 1 M KCl on the mesoporous silica coated ITO electrode with the scan rate of 50 mv/s. (b) The plot of the average number of single molecular spots over three continuous potential sweeps shown in (a) as a function of applied potential from 0 to -1 V. (c) The time derivative of the number of single molecular spots shown in (b) as a function of applied potential. (d) The resorufin concentration dependence of the peak values of the time derivative traces in (c). Inset: linear fit of the first three lower concentrations. S13

14 Figure S12. (a) Distributions of τ on of single resorufin molecules on the mesoporous silica coated ITO surface at potentials from 0 to -1 V. Red lines are the single exponential decay fit, and the corresponding time constants are shown in Fig. 6a in the main text. (b) The residuals from the single exponential decay fit at different potentials in (a). References: (1) Oja, S. M.; Guerrette, J. P.; David, M. R.; Zhang, B. Fluorescence-enabled electrochemical microscopy with dihydroresorufin as a fluorogenic indicator. Anal. Chem. 2014, 86, (2) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications. 2nd ed.; John Wiley: New York, (3) Guerrette, J. P.; Percival, S. J.; Zhang, B. Fluorescence coupling for direct imaging of electrocatalytic heterogeneity. J. Am. Chem. Soc. 2013, 135, S14