Microscopy. Maggie L. Weber, Andrew J. Wilson, and Katherine A. Willets. Corresponding author:

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1 Supporting Information for Characterizing the Spatial Dependence of Redox Chemistry on Plasmonic Nanoparticle Electrodes using Correlated Super-resolution SERS Imaging and Electron Microscopy Maggie L. Weber, Andrew J. Wilson, and Katherine A. Willets Corresponding author: Photoresist Alphanumeric Grid Preparation 2 Covalent Tethering of Nile Blue to Gold Nanoparticles 4 Control Studies for Covalent Tethering of Nile Blue Protocol 5 Electrochemical Well Diagram 9 Proposed Mechanism for Site-specific Redox Potentials based on Potential-Dependent Centroid Changes NB Adsorbed on Gold Colloids 11 Localized Surface Plasmon Control Studies of Aggregates 15 Nanoparticle Aggregate Pairs

2 Photoresist Grid Preparation Shipley Microposit S1818 positive photoresist was spin coated onto the surface of ITO coverslips (SPI Supplies, #1 thickness, Ohm/square) using a Laurell Technologies Corporation spincoater. The spin coating procedure consisted of two steps: step one accelerated the coverslip for 3 seconds to a speed of 500 rpm to evenly spread the photoresist; step two accelerated the coverslip to a speed of 4,000 rpm for 12 seconds to remove excess photoresist. The photoresist-coated coverslip underwent a pre-bake step of approximately 5 minutes at 115 ⁰C. Next, an alphanumeric TEM grid (Ted Pella, Inc.) was placed on top of the photoresist and sandwiched under another clean glass coverslip. This sample was placed in a Karl Süss MA6 Mask Aligner operating in hard contact mode. The substrate with grid atop was exposed for 15 seconds to 365 nm light at ~6.8 mw power. After exposure, the sample was removed from the mask aligner, the TEM grid was carefully moved off the surface and the substrate was developed for 45 seconds in a bath of Microposit MF -319 photoresist developer while gently stirring. Next, a second rinse in a vial of unused developer was carried out for 10 seconds while gently agitating the solution. The slide was then rinsed thoroughly with deionized water and dried under a stream of air. Slides were stored in the dark at atmospheric conditions until usage. Figure S-1 below represents the completed grid formation process. Figure S-2 is an SEM image of the final substrate. Figure S-1. Stepwise representation of the formation of an alphanumeric grid system on an ITO coverslip for correlated electrochemistry experiments using a positive photoresist. 2

3 Figure S-2. SEM image of the non-conductive photoresist grid. Lighter areas correspond to exposed ITO surface. All nanoparticles studied in the electrochemistry experiments were located directly on the ITO. Darker areas represent photoresist remaining on the coverslip after UV exposure and developing. 3

4 Covalent Tethering of Nile Blue to Gold Nanoparticles Scheme S-1. Stepwise functionalization protocol to covalently tether Nile Blue molecule to the surface of gold nanoparticles. Detailed synthetic procedures can be found in main text. 4

5 Control Studies for Covalent Tethering of Nile Blue Protocol The covalent tethering of Nile Blue (NB) to gold nanoparticle surfaces was analyzed by preparing monolayer (or thicker) samples of gold colloids on the electrochemical substrate. By increasing the number of nanoparticles deposited, we achieve a detectable current signal as NB is oxidized and reduced. Nanoaggregate concentrations used in correlated optical/structural studies discussed in the article are too low to produce measurable signals with our potentiostat. As a majority of nanoaggregates discussed within the article were functionalized using the 3- mercaptoproprionic acid (MPA) thiol, we have chosen to focus on control studies on MPAfunctionalized nanoparticle monolayer samples. These samples were prepared by mixing 500 µl of stock 50 nm citrate-capped gold nanoparticles (Nanopartz, A11-50-Citrate) with 200 µl of 5 mm MPA prepared in ethanol. After hours of incubation at room temperature on a rocker plate, the nanoparticles were centrifuged at 14,000 rpm for 10 minutes. The supernatant was removed and the bead was redispersed in an aqueous % w/w citrate solution at ph 11. Next, 25 µl of an aqueous M N-(Dimethylaminopropyl)-N ethylcarbodiimide hydrochloride (EDC, Sigma Aldrich, 98%, 03449) along with 25 µl of M N- hydroxysuccinimide (NHS, Sigma-Aldrich, 98%, 56485) were added to the resuspended thiolated nanoparticles. This sample was vortexed for 30 s and incubated for hours on a rocker plate. After incubation, the sample was centrifuged at 14,000 rpm for 10 minutes. The supernatant was removed and the bead was redispersed in the same % w/w citrate solution at ph 11. In the final step, the NB molecule is covalently tethered via the EDC-NHS coupling linker. A 50 µl aliquot of NB (1 mm) in ethanol was added to the nanoparticles and promptly vortexed for 30 seconds. This solution was left on a rocker plate for hours in the dark to 5

6 allow NB to covalently attach to the particle surface by forming an amide bond upon reaction with the activated EDC-NHS linker. After incubation, the sample was centrifuged at 14,000 rpm for 10 minutes. The supernatant was removed and the bead was redispersed in the same % w/w citrate solution at ph 11. This entire volume of sample (500 µl) was transferred to an electrochemical well and left for hours in a chemical hood to allow the solvent to evaporate, leaving a monolayer (or thicker) of nanoparticles dried on the substrate surface). The well was gently rinsed with nanopure water prior to optical analysis. As there is little clean background area on this substrate, no alignment marker beads were used and therefore we cannot correct any data for stage drift in our optical microscope. 3e-6 25 mv/s 2e-6 Current (A) 1e-6 0-1e Potential (Volts vs. Ag/AgCl) Figure S-3. Cyclic voltammogram of gold colloid monolayer sample with NB tethered via MPA. The potential was scanned at 25 mv/s for 8 complete cycles between ~0 and -0.7 V vs Ag AgCl The cyclic voltammogram in Figure S-3 for this sample reveals a decaying current for both the oxidation and reduction peaks over the course of 8 scans, suggesting that NB molecules are desorbing from the surface. However, we also note that current will be produced in the bulk CV 6

7 from both NB molecules tethered to the gold, as well as any NB molecules non-specifically adsorbed on the ITO. Thus, bulk electrochemical characterization using traditional electrochemical techniques may not be appropriate for characterizing these samples. Figure S-4. Left) EMCCD image of gold colloid monolayer sample under 642 nm illumination with two aggregates labeled, 1 and 2. (Top) SERS Intensity plotted as a function of potential for the entire EMCCD image at left, labeled Bulk Area and the SERS intensity for Aggregates 1 and 2. (Bottom) Intensity timetraces for the EMCCD Bulk Area, Aggregate 1, and Aggregate 2 (from left to right, respectively). Figure S-4 shows the optical data associated with the CV data from Figure S-3. If we integrate over the entire region of interest of the CCD (middle left column), we find the SERS/fluorescence intensity of the NB drops over time, consistent with the decreasing current in Figure S-3 and molecular desorption (we also note that the solution turns blue over the course of these experiments, indicating a high concentration of non-tethered NB). However, if we examine the SERS from individual aggregates (right two columns), we find the intensity is much more stable over multiple scans. This phenomenon is particularly evident in the Aggregate 2 data in Figure S-4, where the SERS intensity plotted as a function of potential and time both remain 7

8 fairly constant compared to the data for Aggregate 1, which shows an intensity decay in the first 200 seconds. In addition to these colloidal monolayer control studies, we have also prepared solutions of the MPA thiol, EDC, sulfo-nhs and NB at the same ratios used to functionalize the gold nanoparticles over a range of ph values (6-12). No nanoparticles were added to these solutions so that the samples could be easily analyzed via electrospray ionization mass spectrometry. Preliminary analysis of these solutions suggests that this coupling reaction proceeds at both ph 6 (typical reaction ph 1, 2 ) and ph 11 (used in this article) as we are able to detect the parent ion for NB covalently tethered to the MPA thiol. These results were collected under low resolution scan settings, yet coupled with the colloidal monolayer control studies, we are confident that we are covalently coupling NB to the gold surface, but cannot confirm the coupling efficiency at this time. We anticipate that some percentage of NB molecules remain in an adsorbed geometry based on the intensity decay that is evident in some aggregate timetraces. 8

9 Electrochemical Well Diagram Figure S-5. Diagram of three electrode cell built with an electrochemical well made from PDMS. Adapted with permission from Wilson, A. J.; Willets, K. A. Visualizing Site-Specific Redox Potentials on the Surface of Plasmonic Nanoparticle Aggregates with Superlocalization SERS Microscopy. Nano Lett., 2014, 14 (2),

10 Proposed Mechanism for Site-specific Redox Potentials based on Potential-Dependent Centroid Changes Figure S-6. Proposed mechanism for interpreting reversible centroid trajectories as a function of applied potential. Unfilled stars represent the reduced, nonemissive form of Nile Blue (black skeletal structure) while filled orange stars represent the oxidized, emissive form of Nile Blue (orange highlighted structure). As the potential is scanned positive (negative), individual molecules are oxidized (reduced), shifting the resulting centroid, as shown by the Gaussian curves and the calculated SERS centroid trajectory in the inset. Reprinted with permission from Wilson, A. J.; Willets, K. A. Visualizing Site-specific Redox Potentials on the Surface of Plasmonic Nanoparticle Aggregates using Superlocalization SERS Microscopy. Nano Lett. 2014, 14 (2), Copyright 2014 American Chemical Society. 10

11 NB Adsorbed on Gold Colloids In addition to studying NB covalently bonded to gold nanoparticle surfaces, numerous experiments were conducted trying to improve adsorption of NB onto colloids. Early studies used colloids at their stock ph value of 7, but these studies produced little if any consistently modulating NB SERS. Based on the matrix studies we completed to optimize EDC coupling, it was discovered that NB-tethered colloids seemed most stable and gave the best NB SERS emission at high ph values. Therefore, we repeated adsorption studies using gold colloids that were centrifuged and resuspended in % citrate at ph 11 prior to adsorbing NB. Typically, 100 µl of stock gold colloids were centrifuged at 7k rpm for 15 minutes. Supernatant was removed and the particle bead was resuspended in the ph 11 citrate solution and then briefly sonicated to redisperse particles. Next 4.2 µl of 1 mm NB in ethanol was added to produce a final concentration of 38.2 µm. These particles were incubated overnight (~15 hours) prior to centrifugation and resuspension in ph 11 citrate solution. Finally, 3 µl of this solution was dropcast onto an ITO coverslip for optical analysis. Figures S-7 and S-8 represent one of the better examples of a gold aggregate showing NB SERS intensity modulation collected in these experiments without EDC coupling. Most aggregates showed strong SERS signals throughout the course of the experiment indicating that adsorption is not only more effective at higher ph, but basic conditions used during the dye incubation step also make it less likely for molecules to desorb later under potential cycling. 11

12 Figure S-7. A) Normalized SERS intensity timetrace showing intensity modulation as a function of the applied potential waveform shown in the middle left column (25 mv/s scan rate). Right columns) Fitted x and y centroid positions. B) SEM image of trimer aggregate that produced this SERS emission. C) Waterfall plot of SERS spectra collected from the trimer during 4 potential cycles. 12

13 . Figure S-8. (A-G) Individual scatter plots of fitted centroid positions for specific potential windows. (0, 0) represents the average position of all centroids collected. The potential range in which these centroid positions were collected is shown above each individual scatter plot. (H) The entire set of centroid positions collected between and V vs. Ag AgCl. The same color coding scheme is used as in plots A-G. 13

14 Unfortunately, we qualitatively noted that intensity modulation of the NB SERS signal is generally worse when NB is adsorbed rather than tethered, likely due to the lack of bonding geometry enabling efficient electron transfer. We also noted that it was much harder to identify site-specific potential trends for the average aggregate in these adsorbed studies. Although 22 gold colloid aggregates were studied for these adsorbed NB experiments, not a single one of them produced a clear potential-dependent trend with regards to emission centroid position. We attribute this to diffusion of molecules across the nanoparticle surface. Ultimately, the tethering protocol proved more effective but only minimal optimization was carried out for these adsorbed studies. With further modification of dye concentration, incubation time, and solvent composition or ph, it may be possible to slow the diffusion process even more. 14

15 Localized Surface Plasmon Control Studies of Aggregates In addition to the electrochemical controls mentioned previously for thiols and EDC coupling, we also tested stability of gold nanoparticles as a function of applied potential. While data may not be available for the exact figures discussed in the article, based on their position in the EMCCD image (as it relates to position of the spectrometer slit within this field of view), we have included numerous examples to indicate that LSPR does not modulate in any way with potential. Minor changes in LSPR intensity are likely due to stage drift of the optical microscope which changes the position of the aggregate in the collection slit of the spectrometer. We have also included SERS spectral data collected for each aggregate. These were collected immediately after collecting LSPR spectra while cycling potential. Note that no potential dependent trend is evident in image B for any of the following three figures (Figures S-9 - S-11). While each aggregate does show some fluctuation in LSPR intensity (Image C in Figures S-9 S-11), there is no correlation to the applied potential as it is cycled between and V. Figure S-11 does display two divots in LSPR intensity (image C, red line) which were due to bubbles floating through the field of view during the experiment. Electrolyte solution is purged prior to analysis but we do note that bubbles occasionally form in the solution. 15

16 Figure S-9. A) SEM image of aggregate analyzed during potential cycling. B) Waterfall plot of the aggregate s LSPR collected over the course of 100 seconds as potential was cycled based on the waveform in C (bottom). C) Top: Integrated intensity of the LSPR peak. Bottom: Potential waveform used while collecting LSPR data for the aggregate in A ( to V versus Ag AgCl, 25 mv/s scan rate, 2 complete cycles). D) Top: SERS intensity timetrace collected for this aggregate while modulating potential based on waveform at bottom ( to V, 25 mv/s scan rate, 4 complete cycles). E) Waterfall plot of NB SERS collecting for the aggregate shown in A, while modulating the potential based on the waveform shown in D (bottom). 16

17 Figure S-10. A) SEM image of aggregate analyzed during potential cycling. B) Waterfall plot of the aggregate s LSPR collected over the course of 100 seconds as potential was cycled based on the waveform in C (bottom). C) Top: Integrated intensity of the LSPR peak. Bottom: Potential waveform used while collecting LSPR data for the aggregate in A ( to V versus Ag AgCl, 25 mv/s scan rate, 2 complete cycles). D) Top: SERS intensity timetrace collected for this aggregate while modulating potential based on waveform at bottom ( to V, 25mV/s scan rate, 4 complete cycles). E) Waterfall plot of NB SERS collecting for the aggregate shown in A, while modulating the potential based on the waveform shown in D (bottom). 17

18 Figure S-11. A) SEM image of aggregate analyzed during potential cycling. Please ignore extraneous text at bottom of SEM image. The aggregate drifted before the image was collected, making it difficult to crop SEM parameters out. B) Waterfall plot of the aggregate s LSPR collected over the course of 100 seconds as potential was cycled based on the waveform in C (bottom). C) Top: Integrated intensity of the LSPR peak. Bottom: Potential waveform used while collecting LSPR data for the aggregate in A ( to V versus Ag AgCl, 25 mv/s scan rate, 2 complete cycles). D) Top: SERS intensity timetrace collected for this aggregate while modulating potential based on waveform at bottom ( to V, 25 mv/s scan rate, 4 complete cycles). E) Waterfall plot of NB SERS collecting for the aggregate shown in A, while modulating the potential based on the waveform shown in D (bottom). 18

19 Nanoparticle Aggregate Pairs Here, we show two examples in Figure S-12 in which two aggregates were present in a single diffraction-limited spot. At the most negative applied potentials (red data), the calculated centroids cluster to a single location, yet as the potential moves towards more positive values (green data), new centroid positions begin to emerge that lie in the direction of the second nanoaggregate. As the potential becomes even more positive (blue and then pink data), the centroid increasingly shifts towards the second nanoaggregate, mapping out a line between the two aggregates. We note that the intermediate centroid points along the line between the two aggregates correspond to the intensity-weighted super-position of centroids associated with the individual aggregates. When both aggregates are SERS-active, the centroid will fall directly between them; however, when one has higher SERS intensity, the centroid will be biased towards that aggregate. In this way, we can map out a complete path between the two. Importantly, these data definitively prove that SERS remains active at more negative potentials on one aggregate within the pair, indicating that NB is more easily oxidized on one nanoparticle aggregate than the other (Figure S-12, red data). 19

20 Figure S-12. NB tethered via MPA for two nanoaggregate pairs (top: 47.6 µm NB; bottom: 38.2 µm NB). Closely spaced aggregates show extreme centroid position modulation in emission centroid location. The most positive applied potential data are shown at left (pink) and the applied potential shifts towards more negative values from left-to-right as indicated by the purple arrow. The centroids associated with the most negative applied potentials (red) are associated with a single aggregate in the pair. Looking at the time traces associated with both sets of aggregate pairs shown in Figure S- 12, we find the familiar reversible centroid (e.g. bowl-shaped ) behavior that we observed in both Figure 2 as well as our previous work (Figures S-13 and S-14). 3 In our previous work, we did not perform correlated nanoparticle structure analysis, so we cannot rule out the possibility that the previous results were due to multiple aggregates present in the same diffraction-limited spot. Nonetheless, we are confident that the results here show both that individual nanoaggregates can have different potentials (as in Figure S-12) and that varying local redox potentials can exist on a single aggregate (as in Figures 4, 7 and 8 from the main text). 20

21 Figure S-13. Timetrace associated with the top example from Figure S-12. (Left) Normalized SERS intensity timetrace showing intensity modulation as a function of the applied potential waveform shown in the middle left column (25 mv/s scan rate). (Right columns) Fitted x and y centroid positions. Figure S-14. Timetrace associated with the bottom example from Figure S-12. (Left) Normalized SERS intensity timetrace showing intensity modulation as a function of the applied potential waveform shown in the middle left column (25 mv/s scan rate). (Right columns) Fitted x and y centroid positions. 21

22 References 1. Li, D.; He, Q.; Cui, Y.; Duan, L.; Li, J., Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability. Biochemical and Biophysical Research Communications 2007, 355, (2), Bartczak, D.; Kanaras, A. G., Preparation of Peptide-Functionalized Gold Nanoparticles Using One Pot EDC/Sulfo-NHS Coupling. Langmuir 2011, 27, (16), Wilson, A. J.; Willets, K. A., Visualizing Site-Specific Redox Potentials on the Surface of Plasmonic Nanoparticle Aggregates with Superlocalization SERS Microscopy. Nano Letters 2014, 14, (2),