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High Resolution Physical Characterization of Single Metallic Nanoparticles

Published: June 28th, 2019



1Physical Measurement Laboratory, National Institute of Standards and Technology, 2Department of Chemical Engineering, Columbia University, 3Department of Applied Physics and Applied Math, Columbia University

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 nanometer-scale pore. The signal is characteristic of the molecule's physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (αHL) 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. 

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 ....

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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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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.  Two chambers 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.......

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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.......

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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 αHL, and for inspiring discussions with Professor Joseph Reiner (Virginia Commonwealth University).


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Name Company Catalog Number Comments
Nanopatch DC System Electronic Biosciences, Inc., EBS
Millipore LC-PAK Millipore vacuum filter
1,2-Diphytanoyl-sn- Glycero-3-Phosphocholine (DPhPC) Avanti Polar Lipids, Alabaster, AL 850356P
Decane, ReagentPlus, ≥99%, Sigma-Aldrich D901
αHL List Biological Laboratories, Campbell, CA
Ag wire Alfa Aesar
2 mm Ag/AgCl disk electrode In Vivo Metric E202
High-impedance amplifier system Electronic Biosciences, San Diego, CA
quartz capillaries
custom polycarbonate test cell
Data Processing and Analysis MOSAIC
Phosphotungstic acid hydrate Sigma-Aldrich 455970
Sodium Chloride Sigma-Aldrich S3014
sodium phosphate monobasic monohydrate Sigma-Aldrich 71507

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