This work was supported by start-up funds from the University of Louisville and funding from Oak Ridge Associated Universities through a Ralph E. Powe Junior Faculty Enhancement Award. The authors thank Dr. Ki-Hyun Cho for creating the image in Figure 1. The metal deposition and SEM were performed at the Micro/Nano Technology Center at the University of Louisville.

1. Gap-mode SERS substrate preparation
<ol>
	<li>Clean No. 1 coverslips (see Table of Materials) using an acetone and water wash, as described below. Perform this step in a cleanroom to ensure that no debris or other unwanted matter is deposited onto the coverslips.
	<ol>
		<li>Place the coverslips in a slide rack. Use tweezers when moving the coverslips/substrates. Place the slide rack in a glass container, and fill it with acetone. 
		CAUTION: Acetone is highly flammable and has...

﻿Analyzing the Heterogeneous Performance of Single Nanoparticles with Electrochemical Methods

Depositing Cu and Ag thin metal films on clean coverslips is vital to ensure that the final film has a roughness no greater than two to four atomic layers (or a root mean square roughness less than or equal to around 0.7 nm). Dust, scratches, and debris present on the coverslip prior to metal deposition are common issues that prevent the fabrication of the smooth film required to produce donut-shaped emission patterns. Hence, it is recommended to sonicate the coverslips in different solvents before the metal deposition a...

Electrochemistry is an important measurement science for studying charge transfer, charge storage, mass transport, etc., with applications in diverse disciplines, including biology, chemistry, physics, and engineering1,2,3,4,5,6,7. Conventionally, electrochemistry involves measurements over an ensemble &#8212;&#160;a&#160;large collection of single entities such as molecules, crystalline domains, nanoparticles, and surface sites. However, understanding how such single entities contribute to ensemble-averaged responses is key for bringing forth new fundamental and mechanistic understandings in chemistry and related fields because of the heterogeneity of electrode surfaces in complex electrochemical environments8,9. For example, ensemble reduction has revealed site-specific reduction/oxidation potentials10, the formation of intermediates and minor catalysis&#160;products11, site-specific reaction kinetics12,13, and charge carrier dynamics14,15. Reducing ensemble averaging is particularly important in improving our understanding beyond model systems to applied systems, such as biological cells, electrocatalysis, and batteries, in which extensive heterogeneity is often found16,17,18,19,20,21,22.
In the past decade or so, there has been an emergence of techniques to study single-entity electrochemistry1,2,9,10,11,12. These electrochemical measurements have provided the capabilities to measure small electrical and ionic currents in several systems and revealed new fundamental chemical and physical characteristics23,24,25,26,27,28. However, electrochemical measurements do not provide information about the identity or structure of molecules or intermediates at the electrode surface29,30,31,32. Chemical information at the electrode-electrolyte interface is central to understanding electrochemical reactions. Interfacial chemical knowledge is typically obtained by coupling electrochemistry with spectroscopy31,32. Vibrational spectroscopy, such as Raman scattering, is well-suited to provide&#160;complementary chemical information on charge transfer and related events in electrochemical systems that predominately utilize, but are not limited to, aqueous solvents30. Coupled with microscopy, Raman scattering spectroscopy provides spatial resolution down to the diffraction limit of light33,34. Diffraction presents a limitation, however, because nanoparticles and active surface sites are smaller in length than optical diffraction limits, which, thus, precludes the study of individual entities35.
Surface-enhanced Raman scattering (SERS) has been demonstrated to be a powerful tool in studying interfacial chemistry in electrochemical reactions20,30,36,37,38. In addition to providing the vibrational modes of reactant molecules, solvent molecules, additives and the surface chemistries of electrodes, SERS provides a signal that is localized to the surface of materials that support collective surface electron oscillations, known as localized surface plasmon resonances. The excitation of plasmon resonances leads to the concentration of electromagnetic radiation at the surface of the metal, thus increasing both the flux of light to and the Raman scattering from surface adsorbates. Nanostructured noble metals such as Ag and Au are commonly used plasmonic materials because they support visible light plasmon resonances, which are desirable for detecting emission with highly sensitive and efficient charge-coupled devices. Although the largest enhancements in SERS come from aggregates of nanoparticles39,40, a new SERS substrate has been developed that allows SERS measurements from individual nanoparticles: gap-mode SERS substrate&#160;(Figure 1)41,42. In gap-mode SERS substrates, a metallic mirror is fabricated and coated with an analyte. Next, nanoparticles are dispersed over the substrate. When irradiated with circularly polarized laser light, a dipolar plasmon resonance formed by the coupling of the nanoparticle and substrate is excited, which enables SERS measurements on single nanoparticles. SERS emission is coupled to the dipolar plasmon resonance43,44,45, which is oriented along the optical axis. With the parallel alignment of the radiating electric dipole and collection optics, only high-angle emission is collected, thus forming distinct donut-shaped emission patterns46,47,48,49 and allowing the identification of single nanoparticles. Aggregates of nanoparticles on the substrate contain radiating dipoles that are not parallel to the optical axis50. In this latter case, low-angle and high-angle emissions are collected and form solid emission patterns46.
Here, we describe a protocol for fabricating gap-mode SERS substrates and a procedure to employ them as working electrodes to monitor electrochemical redox events on single Ag nanoparticles using SERS. Importantly, the protocol using gap-mode SERS substrates allows for the unambiguous identification of single nanoparticles by SERS imaging, which is a key challenge for current methodologies in single nanoparticle electrochemistry. As a model system, we demonstrate the use of SERS to provide a readout of the electrochemical reduction and oxidation of Nile Blue A (NB) on a single Ag nanoparticle driven by a scanning or stepped potential (i.e., cyclic voltammetry, chronoamperometry). NB undergoes a multi-proton, multi-electron reduction/oxidation reaction in which its electronic structure is modulated out of/in resonance with the excitation source, which provides a contrast in the corresponding SERS spectra10,51,52. The protocol described here is also applicable to non-resonant redox-active molecules and electrochemical techniques, which may be pertinent to applications such as electrocatalysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone, microelectronic grade</td>
			<td>J. T. Baker</td>
			<td>9005-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable pipette, Eppendorf Reference 2 5000 mL</td>
			<td>Eppendorf</td>
			<td>4924000100</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance, AB54-S/FACT</td>
			<td>Metter Toledo</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscope, Easy scan 2</td>
			<td>Nanosurf</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>AXXIS Electron Beam Thin Film Deposition System</td>
			<td>Kurt J. Lesker</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Cary 60 UV-Vis Spectrophotometer</td>
			<td>Agilent</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Conductive epoxy, two part</td>
			<td>Electron Microscopy Sciences</td>
			<td>12642-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper pellets, 99.99% pure</td>
			<td>Kurt J. Lesker</td>
			<td>EVMCU40EXE</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper wire, bare, 18 AWG</td>
			<td>VWR</td>
			<td>66248-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Crucible, Graphite E-Beam</td>
			<td>Kurt J. Lesker</td>
			<td>EVCEB-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Scriber</td>
			<td>Ted Pella</td>
			<td>54484</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD Camera, ProEM HS: 1024BX3</td>
			<td>Teledyne Princeton Instruments</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy, Clear</td>
			<td>Gorilla Glue</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Tube Cutter</td>
			<td>Wheeler-Rex</td>
			<td>69012</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Tube, Borossilicate (OD 0.75&#34;, ID 0.62&#34;, L 12&#34;)</td>
			<td>McMaster-Carr</td>
			<td>8729K45</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil, Type-F</td>
			<td>Olympus</td>
			<td>IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope, IX73</td>
			<td>Olympus</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser, Excelsior One 642 nm Free space</td>
			<td>Spectra-Physics</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>LightField</td>
			<td>Teledyne Princeton Instruments</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB 2022b</td>
			<td>MathWorks</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover glass (coverslips), 24&#215;60 mm No. 1</td>
			<td>VWR</td>
			<td>48404-455</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Smartphone Camera Adapter</td>
			<td>qhma</td>
			<td>QHMC017A-S01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nile Blue A, pure</td>
			<td>Acros Organics</td>
			<td>415690100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen, Ultra Pure, Compressed</td>
			<td>Specialty Gases</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective, UPLanXApo 100&#215; Oil Immersion</td>
			<td>Olympus</td>
			<td>14-910</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide Film, Kapton</td>
			<td>3M</td>
			<td>16089-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>VWR</td>
			<td>P285</td>
			<td></td>
		</tr>
		<tr>
			<td>Potentiostat, 660E&#160;</td>
			<td>CH Instruments</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Pt wire</td>
			<td>Alfa Aesar</td>
			<td>10956-BS</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope, Apreo C SEM</td>
			<td>Thermo Fischer Scientific</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>Ted Pella</td>
			<td>16006</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nanoparticles (nanospheres), NanoXact 0.02 mg/mL in 2 mM citrate</td>
			<td>nanoComposix</td>
			<td>AGCN60</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver pellets, 99.99% pure</td>
			<td>Kurt J. Lesker</td>
			<td>EVMAG40EXE-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Rack, Wash-N-Dry</td>
			<td>Diversified Biotech</td>
			<td>WSDR-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Smartphone, iPhone 13 mini</td>
			<td>Apple</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Heptahydrate</td>
			<td>VWR</td>
			<td>0348</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer, IsoPlane SCT320</td>
			<td>Teledyne Princeton Instruments</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Wipers, Light-duty&#160;</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers, KS-04</td>
			<td>Kaisi Hardware</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Utrasonic Generator, sweepSONIK</td>
			<td>Blackstone-NEY Ultrasonics</td>
			<td>809379</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Ultrapurifier, Sartorius Arium mini</td>
			<td>Sartorius</td>
			<td>N.A.</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracking electrochemistry on single nanoparticles with surface-enhanced raman scattering spectroscopy and microscopy

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This nanoscale heterogeneity remains hidden during the ensemble-averaged characterization of nanoparticles. Electrochemical techniques have been developed to measure currents from single nanoparticles but do not provide information about the structure and identity of the molecules that undergo reactions at the electrode surface. Optical techniques such as surface-enhanced Raman scattering (SERS) microscopy and spectroscopy can detect electrochemical events on individual nanoparticles while simultaneously providing information on the vibrational modes of electrode surface species. In this paper, a protocol to track the electrochemical oxidation-reduction of Nile Blue (NB) on single Ag nanoparticles using SERS microscopy and spectroscopy is demonstrated. First, a detailed protocol for fabricating Ag nanoparticles on a smooth and semi-transparent Ag film is described. A dipolar plasmon mode aligned along the optical axis is formed between a single Ag nanoparticle and Ag film. The SERS emission from NB fixed between the nanoparticle and the film is coupled into the plasmon mode, and the high-angle emission is collected by a microscope objective to form a donut-shaped emission pattern. These donut-shaped SERS emission patterns allow for the unambiguous identification of single nanoparticles on the substrate, from which the SERS spectra can be collected. In this work, a method for employing the SERS substrate as a working electrode in an electrochemical cell compatible with an inverted optical microscope is provided. Finally, tracking the electrochemical oxidation-reduction of NB molecules on an individual Ag nanoparticle is shown. The setup and the protocol described here can be modified to study various electrochemical reactions on individual nanoparticles.

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy

Figure 2A shows Ag thin film substrates prepared using an electron beam metal deposition system. The &#34;good&#34; substrate shown in Figure 2A has a homogenous coverage of Ag metal over the glass coverslip, while the &#34;bad&#34; substrate has a non-uniform coverage of Ag. The ultraviolet-visible spectrum of the &#34;good&#34; Ag thin film is shown in Figure 2B, which demonstrates that the film is partially transparent for the vi...

Watch this Scientific Journal Video about Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy at JoVE.com

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy (Video) | JoVE 

The protocol describes how to monitor electrochemical events on single nanoparticles using surface-enhanced Raman scattering spectroscopy and imaging.

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This ...