这项工作得到了路易斯维尔大学的启动资金和橡树岭联合大学通过Ralph E. Powe初级教师增强奖提供的资金支持。作者感谢Ki-Hyun Cho博士创建 图1中的图像。金属沉积和SEM在路易斯维尔大学的微/纳米技术中心进行。

用电化学方法分析单个纳米颗粒的异质性能

提交人声明，他们没有相互竞争的经济利益。

在干净的盖玻片上沉积Cu和Ag薄膜对于确保最终薄膜的粗糙度不超过两到四个原子层（或均方根粗糙度小于或等于约0.7nm）至关重要。在金属沉积之前，盖玻片上存在的灰尘、划痕和碎屑是阻碍制造产生甜甜圈形发射模式所需的光滑薄膜的常见问题。因此，建议在金属沉积之前用不同的溶剂对盖玻片进行超声处理，如果可能的话，在洁净室中进行此过程。此外，应特别注意沉积程序。可能需要清洁?...

电化学是研究电荷转移、电荷存储、质量传递等的重要测量科学，应用于生物学、化学、物理学和工程学等多个学科 1,2,3,4,5,6,7 .传统上，电化学涉及对集合的测量 - 单个实体的大量集合，如分子，晶体结构域，纳米颗粒和表面位点。然而，由于复杂电化学环境中电极表面的异质性，理解这些单一实体如何贡献集成平均响应是化学和相关领域带来新的基本和机理理解的关键8,9。例如，集成还原揭示了位点特异性还原/氧化电位10，中间体和次要催化产物的形成11，位点特异性反应动力学12,13和电荷载流子动力学14,15。减少集成平均对于提高我们对模型系统以外的应用系统的理解尤为重要，例如生物细胞、电催化和电池，其中经常发现广泛的异质性 16,17,18,19,20,21,22。
在过去十年左右的时间里，出现了研究单实体电化学1,2,9,10,11,12的技术。这些电化学测量提供了测量几个系统中小电流和离子电流的能力，并揭示了新的基本化学和物理特性23,24,25,26,27,28。然而，电化学测量不能提供关于电极表面29,30,31,32处分子或中间体的身份或结构的信息。电极-电解质界面处的化学信息是理解电化学反应的核心。界面化学知识通常是通过将电化学与光谱学31,32耦合获得的。振动光谱，如拉曼散射，非常适合提供有关电化学系统中电荷转移和相关事件的补充化学信息，这些系统主要使用但不限于水性溶剂30。与显微镜相结合，拉曼散射光谱提供低至光的衍射极限的空间分辨率33,34。然而，衍射存在局限性，因为纳米颗粒和活性表面位点的长度小于光学衍射极限，因此排除了对单个实体的研究35。
表面增强拉曼散射（SERS）已被证明是研究电化学反应界面化学的有力工具20,30,36,37,38。除了提供反应物分子、溶剂分子、添加剂和电极表面化学的振动模式外，SERS还提供定位于支持集体表面电子振荡的材料表面的信号，称为局部表面等离子体共振。等离激元共振的激发导致电磁辐射集中在金属表面，从而增加光通量和表面吸附物的拉曼散射。纳米结构贵金属如Ag和Au是常用的等离子体材料，因为它们支持可见光等离激元共振，这对于使用高灵敏度和高效的电荷耦合器件检测发射是理想的。尽管SERS的最大增强来自纳米颗粒39,40的聚集体，但已经开发了一种新的SERS衬底，允许从单个纳米颗粒进行SERS测量：间隙模式SERS衬底（图1）41,42。在间隙模式SERS基板中，制造金属镜并涂有分析物。接下来，纳米颗粒分散在基板上。当用圆偏振激光照射时，激发由纳米颗粒和基板耦合形成的偶极等离激元共振，从而能够对单个纳米颗粒进行SERS测量。SERS发射耦合到偶极等离激元共振43,44,45，后者沿光轴定向。通过辐射电偶极子和收集光学器件的平行对准，仅收集高角度发射，从而形成不同的甜甜圈形发射模式46,47,48,49，并允许识别单个纳米颗粒。衬底上的纳米颗粒聚集体含有不平行于光轴50的辐射偶极子。在后一种情况下，收集低角度和高角度发射并形成固体发射模式46。
在这里，我们描述了一种用于制造间隙模式SERS衬底的协议，以及将它们用作工作电极以使用SERS监测单个Ag纳米颗粒上的电化学氧化还原事件的程序。重要的是，使用间隙模式SERS衬底的协议允许通过SERS成像明确识别单个纳米颗粒，这是当前单纳米颗粒电化学方法的关键挑战。作为一个模型系统，我们演示了使用SERS来提供由扫描或阶梯电位（即循环伏安法，计时安培法）驱动的单个Ag纳米颗粒上尼罗河蓝A（NB）的电化学还原和氧化读数。NB经历多质子，多电子还原/氧化反应，其中其电子结构被调制出激发源/与激发源共振，这在相应的SERS光谱10,51,52中提供了对比度。此处描述的协议也适用于非共振氧化还原活性分子和电化学技术，这可能与电催化等应用有关。

<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

研究单个纳米颗粒的电化学反应对于了解单个纳米颗粒的异相性能非常重要。这种纳米级异质性在纳米颗粒的集合平均表征过程中仍然隐藏。已经开发了电化学技术来测量来自单个纳米颗粒的电流，但不提供有关在电极表面发生反应的分子的结构和身份的信息。表面增强拉曼散射（SERS）显微镜和光谱学等光学技术可以检测单个纳米颗粒上的电化学事件，同时提供有关电极表面物种振动模式的信息。本文展示了一种使用SERS显微镜和光谱学跟踪尼罗河蓝（NB）在单个银纳米颗粒上的电化学氧化还原的方案。首先，描述了在光滑和半透明的Ag薄膜上制造Ag纳米颗粒的详细协议。在单个Ag纳米颗粒和Ag薄膜之间形成沿光轴排列的偶极等离激元模式。固定在纳米颗粒和薄膜之间的NB发射的SERS发射耦合到等离激元模式中，高角度发射被显微镜物镜收集，形成甜甜圈状发射图案。这些甜甜圈形的SERS发射模式允许明确识别基板上的单个纳米颗粒，从中可以收集SERS光谱。在这项工作中，提供了一种在与倒置光学显微镜兼容的电化学电池中采用SERS衬底作为工作电极的方法。最后，显示了跟踪单个Ag纳米颗粒上NB分子的电化学氧化还原。可以修改此处描述的设置和协议以研究单个纳米颗粒上的各种电化学反应。

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.

作者聚焦：使用表面增强拉曼散射光谱和显微镜跟踪单个纳米颗粒的电化学  

用表面增强拉曼散射光谱和显微镜跟踪单个纳米颗粒的电化学

A common theme in our research is using light matter interactions and electrical energy to measure, drive, and control interfacial chemical transformations at the nanoscale. In particular, we seek to understand how local environments and reaction intermediates affect selectivity in electric catalysis. Catalytic transformations are traditionally evaluated with ensemble average measurements of the products and the catalyst attributes.The frontier challenges to produce chemical products selectively include reducing this measurement averaging while maintaining the measurement sensitivity, so that we can better understand the actions of individual molecules in the reactive sites of the catalyst. Electrochemical techniques alone do not provide any chemical information on species that form and transform at the electrode surface. Our protocol enables electrochemical measurements at a single nanoparticle using vibrational spectroscopy as a readout, enabling correlations between electrochemical processes and molecular changes.Moving forward, our laboratory will continue to push the boundaries of measurement resolution and space and time to understand chemical and material processes at the single-molecule level and at the timescale of chemical reactions.

图2A 显示了使用电子束金属沉积系统制备的Ag薄膜基板。 图2A 所示的“好”基板在玻璃盖玻片上具有均匀的Ag金属覆盖率，而“坏”基板具有不均匀的Ag覆盖率。“好”Ag薄膜的紫外-可见光谱如图 2B所示，这表明该薄膜对于电磁光谱的可见部分是部分透明的。对于当前协议中用于光谱电化学实验的642 nm激光，“良好”Ag薄膜衬底的?...

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

该协议描述了如何使用表面增强拉曼散射光谱和成像来监测单个纳米颗粒上的电化学事件。

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

我们研究的一个共同主题是使用光物质相互作用和电能来测量、驱动和控制纳米尺度的界面化学转变。特别是，我们试图了解局部环境和反应中间体如何影响电催化中的选择性。传统上，催化转化是通过产品和催化剂属性的集合平均测量来评估的。选择性地生产化学产品的前沿挑战包括在保持测量灵敏度的同时减少这种测量平均值，以便我们能够更好地了解催化剂反应位点中单个分子的作用。单独的电化学技术不能提供有关在电极表面形成和转化的物质的任何化学信息。我们的协议能够使用振动光谱作为读数对单个纳米颗粒进行电化学测量，从而实现电化学过程与分子变化之间的相关性。展望未来，我们的实验室将继续突破测量分辨率、空间和时间的界限，以了解单分子水平和化学反应时间尺度上的化学和材料过程。

tracking-electrochemistry-on-single-nanoparticles-with-surface

Research

JoVE Journal

Chemistry

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

1.2K 次观看.  University of Louisville. 我们研究的一个共同主题是使用光物质相互作用和电能来测量、驱动和控制纳米尺度的界面化学转变。特别是，我们试图了解局部环境和反应中间体如何影响电催化中的选择性。传统上，催化转化是通过产品和催化剂属性的集合平均测量来评估的。选择性地生产化学产品的前沿挑战包括在保持测量灵敏度的同时减少这种测量平均值，以便我们能够更好地了解催化剂反应?...

视频: 作者聚焦：使用表面增强拉曼散射光谱和显微镜跟踪单个纳米颗粒的电化学  

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.

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

科研

化学

Gap-Mode Surface-Enhanced Raman Scattering (SERS) Substrate Preparation

To begin, deposit copper and silver onto the cleaned cover slips using the electron beam thin film deposition system following standard procedures as recommended by the manufacturer. For copper deposition, increase the emission current gradually at 10 milliamperes per minute until the sensor reads a deposition rate close to 10 angstroms per second. Once the desired deposition rate is achieved, close the shutter and set the platen position to zero degrees.Open the shutter to start the deposition process and monitor the thickness on the display of the deposition sensor. Close the shutter when the desired thickness for copper is reached. Then rotate the crucible holder using the knob to direct the beam towards the crucible containing silver pellets, and perform deposition as demonstrated.Next, add 500 microliters of a 50 micromolar Nile Blue solution onto the surface of the silver thin film. After 15 minutes, rinse the silver thin film thoroughly with ultrapure water to remove weakly absorbed Nile Blue molecules. Finally, dry the silver thin film with nitrogen gas.Drop cast 500 microliters of a 100-fold dilution of the silver nanoparticle colloid onto the same region of the silver thin film drop cast with Nile Blue Solution. After 20 minutes, rinse the gap mode surface-enhanced Raman scattering, or SERS substrate, with ultrapure water. Then dry the substrate using nitrogen gas.The ultraviolet visible spectrum of the good silver thin film demonstrates that the film is partially transparent for the visible portion of the electromagnetic spectrum. A representative AFM image of the good substrate is shown here. The variation in the height of the silver thin film substrate is represented by the line profile, demonstrating the uniformity and smoothness of the film.The SEM image of silver nanoparticles drop cast and air dried on a silicon wafer showed an average diameter of approximately 79.2 nanometers.

间隙模式表面增强拉曼散射（SERS）衬底制备

Electrochemical Cell Preparation for Gap-Mode SERS Substrate

To obtain a five-centimeter long glass well, using one hand, firmly grasp the glass tube cutter tool by its handle. On the other hand, hold the glass tube and rotate it continuously so that the wheels in the chain start cutting the glass. Gently squeeze the tool by gradually increasing the pressure on the handles.Use 120 grit sandpaper to smoothen the broken end of the glass well. Then polish with 220 grit sandpaper. To attach the cut glass well to the surface of the substrate, dispense two part epoxy resin onto a small sheet of aluminum foil.Blend the product using a stir stick or a pipette tip. Then, apply the mixture to the bottom rim of the glass well. Glue the glass well to the surface of the gap-mode substrate.Then, apply the remaining mixed product on the outside of the well where it meets the substrate. To attach the electrical connection to the gap-mode SERS substrate, mix two part conductive epoxy resin onto a small sheet of aluminum foil using a five centimeter long copper wire. Attach the wire to the surface of the substrate.

间隙型SERS衬底的电化学电池制备

Bulk Cyclic Voltammetry Measurements of Nile Blue

尼罗河蓝的体循环伏安法测量

Single-Nanoparticle Electrochemical SERS Microscopy and Spectroscopy Measurements

Place the electrochemical cell prepared with the gap mode SERS substrate on the stage of an inverted optical microscope. Secure the substrate to the microscope stage by taping the edges to prevent movement during spectro electrochemical measurements due to the tension of the wires connecting the cell to the potentiostat. Place the silver-silver chloride reference electrode into the home-built stand and fix its position by tightening the screw on the electrode holder stand.Attach the reference electrode to the potentiostat's reference electrode alligator clip. Then attach the platinum wire counter electrode to potentiostat's counter electrode alligator clip. Finally, clip the copper wire attached to the silver film to the potentiostat's working electrode alligator clip.Insert the platinum wire and alligator clip into the electrode holder and tighten the screw to secure its position. Place the electrode holder over the electrochemical cell to insert the electrodes. Then turn on the 642-nanometer laser and adjust the power to 500 microwatts.Next, add a drop of immersion oil to the objective lens. Then, move the focus knob to raise the objective until the oil contacts the bottom of the substrate. Focus the laser onto the surface of the gap mode SERS substrate.After removing one of the eye pieces from the microscope, insert the adapter in its place. Change the mode to video on the camera application and zoom in as much as possible. Scan the gap mode SERS substrate by moving the microscope stage to search for an isolated donut-shaped SERS emission pattern.Once the donut-shaped emission pattern is located, move the light diverter lever of the microscope to direct the admitted light to the spectrometer. Set the grading position to 1000 wave numbers to detect Stokes shifted ramen scattering from 400 to 1600 wave number region. Keeping the laser light focused on the donut-shaped emission pattern, add 3 milliliters of a 0.1-molar phosphate buffer of pH 5 into the electrochemical cell.In the potentiostat's software prepare a cyclic voltammogram experiment with at least three cycles from 0 to minus 0.6 volts versus silver-silver chloride and a scan rate of 50 millivolts per second. Then, run the simultaneous cyclic voltammetry and SERS experiments. Finally, move the light diverter lever, so that the light is directed to the phone camera and start recording a video while running the cyclic voltammetry experiment.Individual silver nanoparticles on the silver thin film can be unambiguously identified by a donut-shaped emission pattern in contrast to a solid emission pattern produced by nanoparticle dimers, trimers, or multimers. The SERS cyclic voltammograms were measured for single nanoparticles, and the Nile blue molecules in and around the gap between the silver nanoparticle and the silver film were electrochemically reduced. Spectroelectrochemical measurements were done with the same applied potential range.Electrochemical modulation of the Nile blue SERS spectrum by stepping the potential causes the peak intensity at the 592 wave number region to decrease with time due to the reduction of the Nile blue molecules. The magnitude of the electric bias altered the reduction kinetics, as evidenced by the decay of the area under the 592 wave number region peak.