We gratefully acknowledge the National Science Foundation (CHE-0847145), and the Henry Dreyfus Teacher-Scholar Awards Program for generously supporting this research. We would like to specifically recognize Christine Davis, Director of Microscopy and Imaging in the Department of Biology at the University of Richmond for her assistance with cross-sectional imaging. Special thanks is given to Drs. T. Leopold, R. Kanters, D. Kellogg, R. Miller, and W. Case, as well as, Russ Collins, Phil Joseph, Carolyn Marks, Mandy Mallory and John Wimbush &#x2013; all of whom make undergraduate research possible at the University of Richmond. A very personal thank you is given to all current, past, and future undergraduate researchers in the Leopold Research Lab.

1. Hexanethiolate Monolayer Protected Gold Clusters Synthesis
Hexanethiolate functionalized monolayer protected gold clusters (MPCs) are synthesized following a 2:1 1-hexanethiol (C6) to gold mole ratio to produce an average structure of Au225(C6)75. 4-9 Speci&#64257;c modi&#64257;cations to the Brust reaction, like ligand type, speci&#64257;c thiol-to-gold ratios, temperature, and reaction delivery rate, or post-synthesis treatments, 9-11 can yield a...

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Protein monolayer electrochemistry is an effective technique used to study interactions between redox proteins and synthetic adsorptive platforms. The effectiveness of this strategy, however, is dependent on the ability to engineer an adsorption interface with a high degree of molecular level control. The MPC-based platforms created by this protocol represent specifically engineered platforms that are able to provide a more homogenous protein adsorption environment3 and facilitate ET over a greater distance<...

<table border="1">
<tr>
<th>Name of the reagent and equipment</th>
<th>Company</th>
<th>Catalogue number</th>
</tr>
<tr>
<td>Tetraoctylammonium bromide</td>
<td>Sigma-Aldrich</td>
<td>294136</td>
</tr>
<tr>
<td>Sodium borohydride</td>
<td>Sigma-Aldrich</td>
<td>213462</td>
</tr>
<tr>
<td>Hydrogen tetrachloroaurate</td>
<td>Sigma-Aldrich</td>
<td>254169-5G</td>
</tr>
<tr>
<td>1-Hexanethiol</td>
<td>Sigma-Aldrich</td>
<td>234192</td>
</tr>
<tr>
<td>Transmission Electron Microscope</td>
<td>JEOL</td>
<td>1010</td>
</tr>
<tr>
<td>Nuclear Magnetic Resonance Spectrometer</td>
<td>Bruker Avance </td>
<td>300 MHz</td>
</tr>
<tr>
<td>Formvar/carbon support film on copper grid (400 mesh)</td>
<td>Electron Microscopy Sciences</td>
<td>FCF400-Cu</td>
</tr>
<tr>
<td>Gold substrate</td>
<td>Evaporated Metal Films Corp.</td>
<td>Custom</td>
</tr>
<tr>
<td>Ag/AgCl Reference electrode</td>
<td>Microelectrodes, Inc.</td>
<td>MI-401F</td>
</tr>
<tr>
<td>Potentiostats </td>
<td>CH Instruments, Inc. </td>
<td>CHI650A, CHI610B</td>
</tr>
<tr>
<td>1,9-Nonanedithiol</td>
<td>Sigma-Aldrich</td>
<td>N29805</td>
</tr>
<tr>
<td>(3-mercaptopropyl)-trimethoxysilane</td>
<td>Sigma-Aldrich</td>
<td>175617</td>
</tr>
<tr>
<td>Ultraviolet Visible Spectrophotometer</td>
<td>Agilent</td>
<td>8453</td>
</tr>
<tr>
<td>Embed 812 epoxy resin</td>
<td>Electron Microscopy Sciences</td>
<td>14120</td>
</tr>
<tr>
<td>&quot;00&quot; BEEM capsule</td>
<td>Electron Microscopy Sciences</td>
<td>70000-B</td>
</tr>
<tr>
<td>Silicon flat mold</td>
<td>Electron Microscopy Sciences</td>
<td>70900</td>
</tr>
<tr>
<td>Diamond knife</td>
<td>Diatome</td>
<td>21-ULE, S12801</td>
</tr>
</table>

synthesis, assembly, and characterization of monolayer protected gold nanoparticle films for protein monolayer electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Watch this Scientific Journal Video about Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry at JoVE.com

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

synthesis-assembly-characterization-monolayer-protected-gold

Research

JoVE Journal

Bioengineering

14.4K Views.  University of Richmond. Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).