High Resolution Physical Characterization of Single Metallic Nanoparticles

Summary

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.

Abstract

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. 

Introduction

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 ....

Protocol

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 National Institute of Standards and Technology, nor does it imply that the materials are the best available.

1. Solution and Ana....

Results

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 immersed in .......

Discussion

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.......

Materials

List of materials used in this article

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	https://pages.nist.gov/mosaic/
Phosphotungstic acid hydrate	Sigma-Aldrich	455970
Sodium Chloride	Sigma-Aldrich	S3014
sodium phosphate monobasic monohydrate	Sigma-Aldrich	71507