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 ...

This nanopore-based detection method has demonstrated the potential to physically characterize and identify individual molecules. Working with metallo-nanoparticles, which is the purpose of this video, is challenging our understanding of what limits the measurement's accuracy and precision. The main advantage of this technique is that this apparatus has greater bandwidth than traditional lipid bilayer memory set-up, which lets us see faster results and more reactions in nanopore.This technique was made possible because in the early 1990s it was shown that gating in protein nanopores, that is, switching between different states, can be controlled and molecules can bind to the pore wall. This technique is being developed for commercial DNA-sequencing applications because it uses minimal samples. It doesn't require fluorescent labels and uses much longer read lengths than conventional methods do.This technique has also been developed to discriminate between polymers based on their size with greater accuracy and speed than gel electrophoresis and chromatography. First, prepare 500 milliliters of a solution of one-molar sodium chloride and 10-millimolar monosodium phosphate in ultrapure water as the electrolyte. It is best to use fresh solutions and samples with this particular set-up.In addition, we found that high-quality deionized water is very important for successfully forming membranes. Next, combine one milligram of lyophilized wild-type monomeric S.aureus alpha hemolysin powder with one milliliter of ultrapure water. Don't forget that working with Staph aureus alpha hemolysin toxin protein can be hazardous.Follow the precautions in the MSDS when performing this procedure. Next, prepare four milliliters of five milligrams per milliliter DPhyPC in endecaine in a glass scintillation vial. Cap the vial with a polytetrafluoroethylene-lined cap and store it at four degrees Celsius for up to a month.After that, dissolve 57.6 milligrams of 12-phosphotungstic acid hydrate in 10 milliliters of the electrolyte to make a two-millimolar polyoxometalate solution. Divide the POM solution into two five-milliliter portions. Use three-molar sodium hydroxide to adjust the pH of one POM solution to 5.5.To begin preparing for the test, assemble the test cell of a planar lipid bilayer electrophysiology apparatus. First, abrade a silver wire with 600-grit sandpaper and soak it in commercially available sodium hypochlorite-based bleach for 10 minutes to make the silver-silver chloride wire electrode. Rinse and dry the wire electrode before inserting it into the quartz capillary of the apparatus.Keep the electrode inside the quartz-nanopore membrane, or QNM, separating the capillary and the reservoir. Then place a cylindrical silver-silver chloride pellet electrode mounted on a silver wire in the reservoir. Connect both electrodes to the amplifier circuit board.Next, open the data acquisition program and start the power supply at zero millivolts. Confirm that no DC current is detected between the electrodes in the empty cell. During this demonstration it's important to watch how the transcapillary pressure can impact membrane formation and subsequent nanopore formation.Without doing these two steps correctly, the experiments won't work. Load about one milliliter of electrolyte into a syringe and connect it to the fluid line connected to the reservoir. Add electrolyte to the reservoir until the face of the QNM is submerged, as indicated by the ionic current between the electrodes saturating the amplifier.If the amplifier does not saturate, unclog the QNM by applying a pop voltage of plus or minus one volt or increasing the capillary pressure by about 300 millimeters of mercury. Use both techniques if necessary. Once the amplifier saturates, withdraw electrolyte from the reservoir until the solution levels drops below the QNM, as indicated by the current returning to zero.To begin the lipid bilayer formation process, raise the electrolyte solution level well above the face of the QNM and note about how much solution that took. Then dip a 10-microliter pipette tip into the DPHyPC solution and draw all visible lipid into the tip. Touch the tip of the pipette to the surface of the electrolyte solution and allow the lipid to flow from the pipette across the air-water interface.Wait two to five minutes for the lipid to spread uniformly across the solution. Next, slowly withdraw the electrolyte until the solution's surface is below the QNM and then slowly add enough electrolyte to raise the solution above the QNM to form the insulating lipid bilayer membrane. If the bilayer does not form after three attempts, apply another portion of lipid as previously described and repeat the process.Once the bilayer seems to have formed, increase the pressure to the capillary to pop the bilayer and then reform it by slowly lowering and raising the solution level in the same way as before. Repeat this process several times to ensure that the QNM is not clogged and a bilayer has formed. Next, thaw an aliquot of alpha hemolysin protein on ice or at room temperature.Fill the reservoir with 250 nanograms of monomeric alpha hemolysin protein and about 250 microliters of ultrapure water. Then, apply a 200 to 400 millivolt bias to induce pore formation. To facilitate pore formation, increase the capillary pressure by 40 to 200 millimeters of mercury to expand the bilayer from the QNM.Once a nanopore forms, reduce the applied bias to the measurement voltage and reduce the pressure to about half of the insertion pressure. To begin the test, readjust the DC-offset voltage to ensure that there is no measured current when the applied potential is set to zero. Next, acquire an ionic current trace for an applied potential of 120 to negative 120 millivolts relative to the reservoir.Confirm that there are no contaminants, which would be indicated by spontaneous current blockades. Add one to six microliters of pH 5.5 two-millimolar POM cluster solution to the reservoir. Apply ionic current time-series measurements at an applied voltage of negative 120 millivolts.The movement of individual anionic phosphotungstic acid molecules through the alpha hemolysin channel produced transient blockades that decreased the mean open-pore ionic current by about 80%The duration and blockade current are both directly related to the particle-pore interaction, allowing ionic species to be differentiated in histograms of the relative blockade depth ratios. At pH 5.5, two peaks were observed with depth ratios of about 0.06 and 0.16. Based on phosphorous NMR, the minor peak was the six-minus phosphotungstate species and the major peak was the seven-minus species.At pH 7.5, the relative concentrations shifted to a higher ratio of the 6-minus species to the seven-minus species. The 20-fold decrease in blockade events relative to pH 5.5 was attributed to degradation to free phosphate and tungstate ions. Fitting the residence-time distributions required multiple exponential functions, indicating multiple types of particle-pore interactions within each ionic species.The shorter residence times at pH 7.5 suggested weaker particle-pore interactions than pH 5.5, consistent with prior studies showing pH-dependent changes in the relative number of fixed charges in or near the alpha hemolysin channel lumen. High-purity deionized water is critical for this particular set-up. A spent organics filter cartridge was the likely cause of our lab being unable to form membranes on the QNMs for months.Individuals new to this particular version of the method might also struggle because the membranes are so small and there is a trick to getting nanopores to form. This method, which was initiated several decades ago, has proven useful for analyzing ions, nucleic acids, and synthetic polymers as well as DNA sequencing. It could also find applications in biophysics and basic cell biology.For example, this procedure can be used to study the physical properties of synthetic polymers and biopolymers to investigate their size, interactions with other molecules, and other important questions.

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Research

JoVE Journal

Chemistry

5.7K visualizações.  National Institute of Standards and Technology. Este método de detecção baseado em nanoporos demonstrou o potencial de caracterizar fisicamente e identificar moléculas individuais. Trabalhar com metallo-nanopartículas, que é o propósito deste vídeo, está desafiando nossa compreensão do que limita a precisão e precisão da medição. A principal vantagem dessa técnica é que este aparelho tem maior largura de banda do que a configuração tradicional de memória bicamada lipídica, o que nos permite ver resultados mais rápidos ...