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.

﻿Analyzing the Heterogeneous Performance of Single Nanoparticles with Electrochemical Methods

The authors declare that they have no competing financial interests.

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><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&quot;, ID 0.62&quot;, L 12&quot;)</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&amp;times;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&amp;times; 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&amp;nbsp;</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&amp;nbsp;</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.

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

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

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

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 ...

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

Research

JoVE Journal

Chemistry

2.3K Views.  University of Louisville. Een gemeenschappelijk thema in ons onderzoek is het gebruik van lichtmaterie-interacties en elektrische energie om interfaciale chemische transformaties op nanoschaal te meten, aan te sturen en te beheersen. In het bijzonder proberen we te begrijpen hoe lokale omgevingen en reactietussenproducten de selectiviteit in elektrische katalyse beïnvloeden. Katalytische transformaties worden traditioneel geëvalueerd met ensemblegemiddelde metingen van de producten en de katalysatorattributen.

Video: Auteur Spotlight: Elektrochemie volgen op enkele nanodeeltjes met oppervlakte-verbeterde Raman-verstrooiingsspectroscopie en microscopie  

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Engineering

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Single-Molecule SERS Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

Author Spotlight: Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas  

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric field of metallic nanostructures. Salt-induced silver nanoparticle aggregation is the most popular method for generating SERS-active substrates; however, it is limited by poor reproducibility, stability, and biocompatibility. The present protocol integrates optical manipulation and SERS detection to develop an efficient analytical platform to address this. A 1064 nm trapping laser and a 532 nm Raman probe laser are combined in a microscope to assemble silver nanoparticles, which generate plasmonic hotspots for in situ SERS measurements in aqueous environments. Without aggregating agents, this dynamic plasmonic silver nanoparticle assembly enables an approximately 50-fold enhancement of the analyte molecule signal. Moreover, it provides spatial and temporal control to form the SERS-active assembly in as low as 0.05 nM analyte-coated silver nanoparticle solution, which minimizes the potential perturbation for in vivo analysis. Hence, this optical trapping-integrated SERS platform holds great potential for efficient, reproducible, and stable molecular analyses in liquids, especially in aqueous physiological environments.

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

The present protocol describes a convenient approach to integrating optical trapping and surface-enhanced Raman spectroscopy (SERS) to manipulate plasmonic nanoparticles for sensitive molecular detection. Without aggregating agents, the trapping laser assembles plasmonic nanoparticles to enhance the SERS signals of target analytes for in situ spectroscopic measurements.

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric ...

Bioengineering

Label-Free SERS Analysis: Au@Carbon Dot Nanoprobes for Molecular Fingerprinting

Label-Free Surface-Enhanced Raman Scattering Bioanalysis Based on Au@Carbon Dot Nanoprobes

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular fingerprint information of biological samples, as well as its potential in single-cell analysis. This work aims to establish a simple strategy for label-free SERS bioanalysis based on Au@carbon dot nanoprobes (Au@CDs). Here, polyphenol-derived CDs are utilized as the reductant to rapidly synthesize core-shell Au@CD nanostructures, which allows powerful SERS performance even when the concentration of methylene blue (MB) is as low as 10-9 M, due to the cooperative Raman enhancement mechanism. For bioanalysis, Au@CDs can serve as a unique SERS nanosensor to identify the cellular components of biosamples (e.g., cancer cells and bacteria). The molecular fingerprints from different species can be further distinguished after combination with the principal component analysis. In addition, Au@CDs also enable label-free SERS imaging to analyze intracellular composition profiles. This strategy offers a feasible, label-free SERS bioanalysis, opening up a new prospect for nanodiagnosis.

Author Spotlight: Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging 

In this study, we developed a low-cost surface-enhanced Raman scattering (SERS)-based fingerprint nanoprobe with favorable biocompatibility to show label-free live cell bioimaging and detect two bacterial strains, showing in detail how to get SERS spectra of living cells in a non-destructive method.

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular ...

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

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