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 diverse range of MPCs with varying core sizes and functional protective groups, respectively.4 The MPC approximate (average) compositions of MPCs functionalized with various alkanethiol groups can be determined by proton (1H) nuclear magnetic resonance (NMR) analysis of iodine-decomposed samples.
<ol>
	<li>Dissolve 1.1 g tetraoctylammonium bromide (TOABr) in 30 mL of toluene with appropriate fume hood ventilation.</li>
	<li>Dissolve 0.38 g sodium borohydride (NaBH4) in &tilde;20 mL of 18 M&Omega; ultrapuri&#64257;ed water (UP H2O) and allow to chill over ice for at least 30 min.</li>
	<li>Dissolve 0.31 g hydrogen tetrachloroaurate (HAuCl4) in &tilde;20 mL of UP H2O and quantitatively transfer the solution mixture to the TOABr-toluene solution using an additional &tilde;5 mL of UP H2O in order to phase-transfer the aqueous gold solution to the nonaqueous solution. Stir rigorously while lightly capped for 30 min so the burnt orange aqueous and clear nonaqueous phases are mixing well.</li>
	<li>Transfer both the clear aqueous and burnt orange nonaqueous phases to a separatory funnel. Discard the aqueous (bottom) layer and decant the nonaqueous (top) layer into a clean flask.</li>
	<li>Add C6 in a ratio of 2:1 with HAuCl4 to the nonaqueous solution. Stir for 30 min to form a Au(I) polymer, as detected by a color change from reddish orange to a pale yellow, nearly colorless solution.</li>
	<li>Transfer the reaction mixture to an insulated ice bath and chill to 0&deg;C for at least 30 min with stirring.</li>
	<li>Quantitatively and quickly add the chilled NaBH4 solution to the reaction mixture in order to reduce Au(I) to a metallic gold in the presence of thiols, instantaneously forming a thick black solution of MPCs upon addition. Stir reaction overnight at 0&deg;C.</li>
	<li>Transfer the reaction mixture to a separatory funnel, discard the aqueous (bottom) layer into a waste beaker, and rotary evaporate the nonaqueous (top) toluene layer to near complete dryness leaving a heavy black sludge in the flask.</li>
	<li>Precipitate the MPCs by adding acetonitrile and allowing to sit overnight.</li>
	<li>Collect MPCs by vacuum filtration using a glass frit of medium porosity with rubber fittings and side-armed flask with aspirator and rinse with a copious amount of acetonitrile.</li>
	<li>Allow MPCs to air dry, weigh product, characterize by transmission electron microscopy (TEM) and NMR analysis, and store capped for future use. Obtain TEM images by drop-casting MPCs dissolved in toluene onto formvar/carbon support film on copper grid (400 mesh) and operating the TEM instrument at 80-100 kV. The average core size can be estimated using image analysis software such as Image J (freeware).</li>
</ol>
2. Film Assembly: Dithiol-linked MPC Film Assembly for Protein Monolayer Electrochemistry
The gold substrate is first electrochemically cleaned and modified with a C6 SAM before immersing in alternating solutions of dithiol linking molecules and C6 modified MPCs to make up a "dip cycle," which is repeated several times to ultimately form a dithiol-linked MPC film assembly. As described in prior studies,2 the original plasmid for the Pseudomonas aeruginosa azurin (AZ) protein was graciously given by Dr. Corey Wilson of Rice University and AZ was provided as a purified and lyophilized powder by the University of Richmond professor, Dr. Jonathan Dattelbaum, that was subsequently rehydrated with 4.4 mM potassium phosphate buffer (KPB, pH = 7.0, &mu; = 10 mM) to create a 5-10 &mu;M solution as verified by ultraviolet visible (UV-Vis) analysis.
<ol>
	<li>Assemble the electrochemical (echem) sandwich cell in the following order from bottom to top: first Lucite retainer plate, gold substrate as a working electrode, brass electrical contact to gold working electrode, first rubber gasket, Viton o-ring that defines the electrode area (0.32 cm2), glass cell body, second rubber gasket, and second Lucite retainer plate. The entire cell is held together by threaded rods and carefully tightened wing nuts. The cell is fitted with a commercially purchased reference electrode that houses a glass barrel with 1 M saturated KCl, Ag/AgCl reference wire, and a Pt auxiliary electrode wire.</li>
	<li>Electrochemically clean the gold substrate by performing cyclic voltammetry (CV) in the potential windows from 0.2 to 0.9 V, 0.2 to 1.2 V, and 0.2 to 1.35 V (versus Ag/AgCl, KCl) at 100 mV/s in a solution of 0.1 M H2SO4 and 0.01 M KCl.</li>
	<li>Measure the charging current of the cleaned bare gold substrate by performing CV at "standard conditions," including a potential window from 0.1 to 0.4 V (versus Ag/AgCl, KCl) scanned at 100 mV/s in KPB.1 Discard KPB and rinse successively with UP H2O, ethanol (EtOH), UP H2O, and EtOH.</li>
	<li>Expose the cleaned gold substrate to &tilde;300 &mu;l of 5 mM C6 solution in EtOH and allow to sit overnight to form an ordered C6 SAM. Discard C6 solution from the cell and rinse successively with EtOH, UP H2O, EtOH, and UP H2O.</li>
	<li>Measure the charging current of the SAM at standard conditions. Discard KPB and rinse successively with UP H2O, EtOH, UP H2O, and EtOH. The charging current should be markedly decreased from that of the bare gold measurement (step 2.3).1</li>
	<li>Expose the SAM modified gold substrate to &tilde;300 &mu;l of 5 mM 1,9-nonanedithiol (NDT) solution in EtOH and allow to sit for 1 hr to interdisperse NDT linking molecules within the C6 SAM. Discard NDT solution and rinse successively and thoroughly with EtOH, UP H2O, EtOH, UP H2O, and methylene chloride (CH2Cl2).</li>
	<li>Expose the gold substrate to a MPC solution of CH2Cl2 (&tilde;1 mg/mL) with agitation by slowly bubbling with N2 gas for 1 hr. If necessary, replace evaporated MPC solution with more CH2Cl2. This is the anchoring MPC layer of the film assembly. Discard MPC solution and again rinse successively with CH2Cl2, UP H2O, and KPB.</li>
	<li>Measure the charging current of the MPC layer at standard conditions. Discard KPB and rinse successively with UP H2O and CH2Cl2.</li>
	<li>Expose the gold substrate to &tilde;300 &mu;l of 5 mM NDT solution of CH2Cl2 with agitation by slowly bubbling with N2 gas for 20 min.</li>
	<li>Discard NDT and rinse thoroughly with CH2Cl2. Repeat steps 2.7 and 2.8 to deposit the second MPC layer of the film assembly.</li>
	<li>To deposit additional MPC layers, steps 2.9 and 2.10 are repeated. With each additional MPC layer a corresponding increase in the charging current is observed.</li>
	<li>After the networked MPC film is complete, rinse the film modified substrate with KPB. AZ protein is adsorbed on the MPC film assembly by injecting &tilde;150 &mu;L of &tilde;5-10 &mu;M AZ solution of KPB into the echem sandwich cell and allowing to sit capped and refrigerated for at least 1 hr.</li>
	<li>Remove the echem cell from the refrigerator and allow it to return to near room temperature. Thoroughly rinse with KPB, refill echem cell with KPB, and bubble KPB with N2 gas for 10 min.</li>
	<li>Protein monolayer electrochemical studies are performed as CV in the potential window from -0.25 V to 0.25 V (versus Ag/AgCl, KCl) scanned at 100 mV/sec in KPB.</li>
</ol>
3. Film Assembly: Dithiol-linked MPC Film Assembly for Optical Tracking
Prior to growing MPC films for optical evaluation, glass slide sections are pre-cleaned with Piranha solution (CAUTION! 2:1 concentrated H2SO4 and H2O2) and treated with (3-mercaptopropyl)-trimethoxysilane (3-MPTMS).1-2 MPC films are then assembled on these modified glass slides using the "dip cycle" technique as previously described above.
<ol>
	<li>Rinse a 3-MPTMS modified glass slide with CH2Cl2 and place it in a MPC solution of CH2Cl2 (&tilde;1 mg/mL) for 1 hr while agitating on a shaker at low speed. This completes the first MPC layer of the film assembly by anchoring MPCs to the mercaptans endgroups of the silane. Rinse the slide thoroughly with CH2Cl2, and dry with N2 gas. Take a UV-Vis spectrum (from 400 to 1000 nm) of the slide, and rinse again with CH2Cl2.</li>
	<li>Place the slide in a 5 mM NDT solution of CH2Cl2 for 1 hr while agitating on a shaker at low speed. Rinse the slide with CH2Cl2.</li>
	<li>Place the slide in a MPC solution for 1 hr while agitating on a shaker at low speed. This completes the second MPC layer of the film assembly. Rinse the slide thoroughly with CH2Cl2, dry with N2 gas, and take a UV-Vis spectrum (from 400 to 1000 nm) of the slide. The absorbance across the spectrum should be increasing as additional MPC layers are adsorbed to the film assembly.</li>
	<li>To deposit additional MPC layers, steps 3.2-3.3 are repeated.</li>
</ol>
4. Characterization of Monolayer Protected Gold Cluster Film Assemblies by Cross Sectional Transmission Electron Microscopy
TEM cross sections are prepared by re-embedding en face embedded films.2, 12 This is done by first attaching a MPC film assembled on a 3-MPTMS modified glass slide onto a clean, standard microscope slides using Embed 812 epoxy resin to allow for improved handling during the procedure below. Use caution with the applied heat as higher temperatures will decompose the MPCs within the film.
<ol>
	<li>Mix Embed 812 epoxy resin and allow to thicken for at least 12 hr.</li>
	<li>Fill a "00" BEEM capsule with epoxy resin and invert on top of the MPC film sample (prepared in section 3). Place pressure on the capsule so that a bubble rises to the top of capsule, creating a seal between the epoxy resin and MPC film sample. Allow to polymerize for at least 18 hr at 60&deg;C and then cool the mounted slides to room temperature.</li>
	<li>Heat mounted slides for 20 sec on a cast aluminum hot plate at 200&deg;C in order to facilitate the removal of the block with attached MPC en face film.</li>
	<li>Cut the sample containing the film off of the BEEM capsule block using a jeweler's saw.</li>
	<li>Re-embed the removed portion in a silicon flat mold with the MPC film side up facing the interior of the silicon well. Fill the silicon well with epoxy resin at room temperature and allow to polymerize for at least 18 hr at 60&deg;C. Cool the sample to room temperature.</li>
	<li>Obtain thin sample sections of 60-80 nm on a Leica UCT ultramicrotome by using a diamond knife to cut sections perpendicular to the knife's edge.</li>
	<li>Place sliced sections on formvar/carbon support film on copper grid (400 mesh) and take TEM images of prepared cross sections of MPC film assemblies.</li>
</ol>
5. Representative Results:
<img alt="Figure 1" src="/files/ftp_upload/3441/3441fig1.jpg" /> 
Figure 1 Double layer charging current monitoring of MPC film growth for a total of 5 dipping cycles (alternating exposure to MPC and NDT solutions). Charging current increased systematically with each dipping cycle, adding "layers" of MPC to the film (Fig. 2). The cyclic voltammograms were collected using a potential window from 0.1 to 0.4 V (versus Ag/AgCl, KCl) scanned at 100 mV/s in 4.4 mM potassium phosphate buffer (pH = 7.0, &mu; = 10 mM). Reprinted with permission from M.L. Vargo, C.P. Gulka, J.K. Gerig, C.M. Manieri, J.D. Dattelbaum, C.B. Marks, N.T. Lawrence, M.L. Trawick, and M.C. Leopold, Langmuir 26(1), 560-569. Copyright 2010 American Chemical Society.
<img alt="Figure 2" src="/files/ftp_upload/3441/3441fig2.jpg" /> 
Figure 2 (a) Schematic representation of AZ protein adsorbed to a dithiol-linked MPC film assembly. (b) Typical cyclic voltammogram for AZ adsorbed to MPC film assembly collected using a potential window from -0.25 to +0.25 V (versus Ag/AgCl, KCl) scanned at 100 mV/s in 4.4 mM potassium phosphate buffer (pH = 7.0, &mu; = 10 mM).
<img alt="Figure 3" src="/files/ftp_upload/3441/3441fig3.jpg" /> 
Figure 3 Representative UV-Vis spectral monitoring of a dithiol-linked MPC film growth on a 3-MPTMS modified glass slide. A dip cycle consists of an exposure of the glass slide to NDT linker solution followed an exposure to MPC solution. Each subsequent dip results in growth in film thickness and a concurrent absorbance increase. As the number of dip cycles increases, the surface plasmon band is gradually defined at &tilde;520 nm. Reprinted with permission from M.L. Vargo, C.P. Gulka, J.K. Gerig, C.M. Manieri, J.D. Dattelbaum, C.B. Marks, N.T. Lawrence, M.L. Trawick, and M.C. Leopold, Langmuir 26(1), 560-569. Copyright 2010 American Chemical Society.
<img alt="Figure 4" src="/files/ftp_upload/3441/3441fig4.jpg" /> 
Figure 4 Transmission electron microscopy (TEM) cross sectional image analysis of a dithiol-linked MPC film assembly. Inset: Typical TEM image of hexanethiolate functionalized MPCs used in the film assembly. TEM analysis determined an average gold core diameter of the MPCs to be &tilde;2 nm using Image J analysis. Reprinted with permission from M.L. Vargo, C.P. Gulka, J.K. Gerig, C.M. Manieri, J.D. Dattelbaum, C.B. Marks, N.T. Lawrence, M.L. Trawick, and M.C. Leopold, Langmuir 26(1), 560-569. Copyright 2010 American Chemical Society.

<ol>
	<li>Loftus, A. F., Reighard, K. P., Kapourales, S. A., Leopold, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer-Protected+Nanoparticle+Film+Assemblies+as+Platforms+for+Controlling+Interfacial+and+Adsorption+Properties+in+Protein+Monolayer+Electrochemistry.">Monolayer-Protected Nanoparticle Film Assemblies as Platforms for Controlling Interfacial and Adsorption Properties in Protein Monolayer Electrochemistry.</a> J. Am. Chem. Soc. 130, 1649-1661 (2008).</li><li>Vargo, M. L., Gulka, C. P., Gerig, J. K., Manieri, C. M., Dattelbaum, J. D., Marks, C. B., Lawrence, N. T., Trawick, M. L., Leopold, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distance+Dependence+of+Electron+Transfer+Kinetics+for+Azurin+Protein+Adsorbed+to+Monolayer+Protected+Nanoparticle+Film+Assemblies.">Distance Dependence of Electron Transfer Kinetics for Azurin Protein Adsorbed to Monolayer Protected Nanoparticle Film Assemblies.</a> Langmuir. 26, 560-569 (2010).</li><li>Doan, T. T., Vargo, M. L., Gerig, J. K., Gulka, C. P., Trawick, M. L., Dattelbaum, J. D., Leopold, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+analysis+of+azurin+thermodynamic+and+adsorption+properties+at+monolayer-protected+cluster+film+assemblies+-+Evidence+for+a+more+homogeneous+adsorption+interface.">Electrochemical analysis of azurin thermodynamic and adsorption properties at monolayer-protected cluster film assemblies - Evidence for a more homogeneous adsorption interface.</a> Journal of Colloid and Interface Science. 352, 50-58 (2010).</li><li>Brust, M., Walker, M., Bethell, D., Schriffrin, D. J., Whyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+Thiol-derivatised+Gold+Nanoparticles+in+a+Two-phase+Liquid-Liquid+System.">Synthesis of Thiol-derivatised Gold Nanoparticles in a Two-phase Liquid-Liquid System.</a> J. Chem. Soc. Chem. Commun. , 801-802 (1994).</li><li>Scouten, W. H., Luong, J. H., Brown, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+or+protein+immobilization+techniques+for+applications+in+biosensor+design.">Enzyme or protein immobilization techniques for applications in biosensor design.</a> Trends Biotechnol. 13, 178-185 (1995).</li><li>Davis, J., Vaughan, D. H., Cardosi, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+of+biosensor+construction.">Elements of biosensor construction.</a> Enzyme Microb. Technol. 17, 1030-1035 (1995).</li><li>Ramsay, G., Ed, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Commercial+Biosensors+-+Applications+to+Clinical,+Bioprocess,+and+Environmental+Samples.">In Commercial Biosensors - Applications to Clinical, Bioprocess, and Environmental Samples.</a> , (1998).</li><li>Ghindilis, A. L., Atanasov, P., Wilkins, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosensors:+electrochemical+sensing+and+other+engineering+approaches.">Immunosensors: electrochemical sensing and other engineering approaches.</a> Biosens. Bioelectron. 13, 113-131 (1998).</li><li>Templeton, A. C., Wuelfing, W. P., Murray, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer-Protected+Cluster+Molecules.">Monolayer-Protected Cluster Molecules.</a> Accounts. Chem. Res. 33, 27-36 (2000).</li><li>Aguila, A., Murray, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer-Protected+Clusters+with+Fluorescent+Dansyl+Ligands.">Monolayer-Protected Clusters with Fluorescent Dansyl Ligands.</a> Langmuir. 16, 5949-5954 (2000).</li><li>Hostetler, M. J., Templeton, A. C., Murray, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Place-Exchange+Reactions+on+Monolayer-Protected+Gold+Cluster+Molecules.">Dynamics of Place-Exchange Reactions on Monolayer-Protected Gold Cluster Molecules.</a> Langmuir. 15, 3782-3789 (1999).</li><li>Wanunu, M., Popovitz-Biro, R., Cohen, H., Vaskevich, A., Rubenstein, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination-Based+Gold+Nanoparticle+Layers.">Coordination-Based Gold Nanoparticle Layers.</a> J. Am. Chem. Soc. 127, 9207-9215 (2005).</li><li>Gaylean, A. A., Day, R. W., Malinowski, J., Kittredge, K. W., Leopold, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte-linked+film+assemblies+of+nanoparticles+and+nanoshells:+Growth,+stability,+and+optical+properties.">Polyelectrolyte-linked film assemblies of nanoparticles and nanoshells: Growth, stability, and optical properties.</a> Journal of Colloid and Interface Science. 331, 532-542 (2009).</li><li>Campbell-Rance, D. S., Doan, T. T., Leopold, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweep,+Step,+Pulse,+and+Frequency-Based+Techniques+Applied+to+Protein+Monolayer+Electrochemistry+at+Nanoparticle+Interfaces.">Sweep, Step, Pulse, and Frequency-Based Techniques Applied to Protein Monolayer Electrochemistry at Nanoparticle Interfaces.</a> , .</li></ol>

No conflicts of interest declared.

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

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JoVE Journal

Bioengineering

14.4K 次观看.  University of Richmond. Alkanethiolate稳定的黄金被称为单层保护集群（储值卡）胶体合成，特点，并组装成薄膜作为一个简单的氧化还原蛋白的蛋白质单层的电化学像吸附接口绿脓杆菌阿苏林（AZ）和细胞色素 C（CYT C）。

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

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...