Name: high resolution physical characterization of single metallic nanoparticles
Uploaded: 2019-06-28T11:00:00
Description: Watch this Scientific Journal Video about High Resolution Physical Characterization of Single Metallic Nanoparticles at JoVE.com

We are grateful for financial support from the European Molecular Biology Organization for a postdoctoral fellowship (to J.E.) and a grant from the NIH NHGRI (to J.J.K.). We appreciate the help of Professors Jingyue Ju and Sergey Kalachikov (Columbia University) for providing heptameric &#945;HL, and for inspiring discussions with Professor Joseph Reiner (Virginia Commonwealth University).

Note: The protocol below is specific to the Electronic BioSciences (EBS) Nanopatch DC System. However, it can be readily adapted to other electrophysiology apparatus used to measure the current through planar lipid bilayer membranes (standard lipid bilayer membrane chamber, U-tube geometry, pulled microcapillaries, etc.). The identification of commercial materials and their sources is given to describe the experimental results. In no case does this identification imply recommendation by the Nati...

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T., Hill, C. L., Judd, D. A., Schinazi, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyoxometalates+in+medicine.">Polyoxometalates in medicine.</a> Chemical Reviews. 98 (1), 327-358 (1998).</li><li>Gao, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition-metal-substituted+polyoxometalate+derivatives+as+functional+anti-amyloid+agents+for+Alzheimer's+disease.">Transition-metal-substituted polyoxometalate derivatives as functional anti-amyloid agents for Alzheimer's disease.</a> Nature Communications. 5, 3422 (2014).</li><li>Pope, M. 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Due to their anionic charge, POMs likely associate with organic counter cations through electrostatic interactions. Therefore, it is important to identify the proper solution conditions and the right electrolyte environments (especially cations in solution) to avoid complex formation with POMs. Particular care is required in the buffer choice. For example, the capture rate of POMs with tris(hydroxymethyl)aminomethane and citric acid-buffered solutions is significantly lower than that in phosphate buffered solution, likel...

Detecting biomolecular analytes at the single molecule level can be performed by using nanopores and measuring ionic current modulations. Typically, nanopores are divided into two categories based on their fabrication: biological (self-assembled from protein or DNA origami)1,2,3, or solid-state (e.g., manufactured with semiconductor processing tools)4,5. While solid-state nanopores were suggested as potentially more physically robust and can be used over a wide range of solution conditions, protein nanopores thus far offer greater sensitivity, more resistance to fouling, greater bandwidth, better chemical selectivity, and a greater signal to noise ratio.
A variety of protein ion channels, such as the one formed by Staphylococcus aureus &#945;-hemolysin (&#945;HL), can be used to detect single molecules, including ions (e.g., H+ and D+)2,3, polynucleotides (DNA and RNA)6,7,8, damaged DNA9, polypeptides10, proteins (folded and unfolded)11, polymers (polyethylene glycol and others)12,13,14, gold nanoparticles15,16,17,18,19, and other synthetic molecules20.
We recently demonstrated that the &#945;HL nanopore can also easily detect and characterize metallic clusters, polyoxometalates (POMs), at the single molecule level. POMs are discrete nanoscale anionic metal oxygen clusters that were discovered in 182621, and since then, many more types have been synthesized. The different sizes, structures, and elemental compositions of polyoxometalates that are now available led to a wide range of properties and applications including chemistry22,23, catalysis24, material science25,26, and biomedical research27,28,29.
POM synthesis is a self-assembly process typically carried out in water by mixing the stoichiometrically required amounts of monomeric metal salts. Once formed, POMs exhibit a great diversity of sizes and shapes. For example, the Keggin polyanion structure, XM12O40q- is composed of one heteroatom (X) surrounded by four oxygens to form a tetrahedron (q is the charge). The heteroatom is centrally located within a cage formed by 12 octahedral MO6 units (where M = transition metals in their high oxidation state), which are linked to one another by neighboring shared oxygen atoms. While tungsten polyoxometalates structure is stable in acidic conditions, hydroxide ions lead to the hydrolytic cleavage of metal-oxygen (M-O) bonds30. This complex process results in the loss of one or more MO6 octahedral subunits, leading to the formation of monovacant and trivacant species and eventually to the complete decomposition of the POMs. Our discussion here will be limited to the partial decomposition products of 12-phosphotungstic acid at pH 5.5 and 7.5.
The goal of this protocol is to detect discrete metal oxygen clusters at the single molecule limit using a biological nanopore-based electronic platform. This method allows the detection of metallic clusters in solution. Multiple species in solution can be discriminated with greater sensitivity than conventional analytical methods33. With it, subtle differences in POM structure can be elucidated, and at concentrations markedly lower than those required for NMR spectroscopy. Importantly, this approach even allows the discrimination of isomeric forms of Na8HPW9O341.

<table>
	<tbody>
		<tr>
			<td>Nanopatch DC System</td>
			<td>Electronic Biosciences, Inc., EBS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore LC-PAK</td>
			<td>Millipore vacuum filter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-Diphytanoyl-sn- Glycero-3-Phosphocholine (DPhPC)</td>
			<td>Avanti Polar Lipids, Alabaster, AL</td>
			<td>850356P</td>
			<td></td>
		</tr>
		<tr>
			<td>Decane, ReagentPlus, &#8805;99%,</td>
			<td>Sigma-Aldrich</td>
			<td>D901</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;HL</td>
			<td>List Biological Laboratories, Campbell, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ag wire</td>
			<td>Alfa Aesar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mm Ag/AgCl disk electrode</td>
			<td>In Vivo Metric</td>
			<td>E202</td>
			<td></td>
		</tr>
		<tr>
			<td>High-impedance amplifier system</td>
			<td>Electronic Biosciences, San Diego, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>quartz capillaries</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>custom polycarbonate test cell</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data Processing and Analysis MOSAIC</td>
			<td>https://pages.nist.gov/mosaic/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphotungstic acid hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>455970</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate monobasic monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>71507</td>
			<td></td>
		</tr>
	</tbody>
</table>

high resolution physical characterization of single metallic nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Over the past two decades, membrane-bound protein nanometer-scale pores have been demonstrated as versatile single-molecule sensors. Nanopore-based measurements are relatively straightforward to execute.&#160; Two chambers&#160;filled with electrolyte solution are separated by a nanopore embedded in an electrically insulating lipid membrane. Either a patch-clamp amplifier or an external power supply provides an electrostatic potential across the nanopore via Ag/AgCl electrodes im...

Watch this Scientific Journal Video about High Resolution Physical Characterization of Single Metallic Nanoparticles at JoVE.com

High Resolution Physical Characterization of Single Metallic Nanoparticles

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

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