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 National Institute of Standards and Technology, nor does it imply that the materials are the best available.
1. Solution and Analyte Preparation
<ol>
	<li>Prepare all electrolyte solutions with 18 M&#937;-cm water from a Type-1 water purification system to remove trace organic species and then filter all electrolyte solutions through a 0.22 &#956;m vacuum filter immediately before ion channel recordings. 
	Note: The water quality is a critical factor for the stability and longevity of the membrane nanopore system.</li>
	<li>Wild Type &#945;HL.
	<ol>
		<li>Follow MSDS precautions when handling the &#945;HL toxin protein.</li>
		<li>Mix lyophilized wild-type monomeric S. aureus &#945;-Hemolysin (&#945;HL) powder with 18 M&#937;-cm water at 1 mg/mL. Distribute 10 to 30 &#956;L aliquots of the sample into cryo-safe centrifuge tubes, quickly flash freeze in liquid nitrogen and then store at -80 &#176;C. Alternatively, use purified preformed heptameric &#945;HL31.</li>
	</ol>
	</li>
	<li>Dissolve the lipid 1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine (DPhyPC) to 0.2 mg/mL in n-decane in a 4 mL glass scintillation vial with a polytetrafluoroethyleneeflon-coated cap. Store the solution at 4 &#176;C for repeated use for up to one month.</li>
	<li>Prepare phosphotungstic acid solutions.
	<ol>
		<li>Follow MSDS precautions when handling the&#160;phosphotungstic acid&#160;powder and prepare a 2 mM phosphotungstic acid stock solution by dissolving 57.6 mg of H3PW12O40 into 10 mL of a 1 M NaCl and 10 mM NaH2PO4 solution, which constitutes the stock solution.</li>
		<li>Take 5 mL of this solution and adjust the pH to 5.5 with 3 M NaOH. Adjust the pH of the other 5 mL of the stock solution to 7.5 with 3 M NaOH. 
		Note: At pH 5.5, 12-phosphotungstic acid (PTA, H3PW12O40) decomposes primarily into the monovacant anion [PW11O39]7-.</li>
	</ol>
	</li>
</ol>
2. Test Cell Assembly
<ol>
	<li>Assemble the test cell per the manufacturer&#8217;s instructions.</li>
	<li>Soak one Ag/AgCl wire in bleach (sodium hypochlorite) for 10 minutes after abrading it with 600 grit sandpaper. Position the electrode inside the quartz nanopore membrane (QNM).</li>
	<li>Place a cylindrical AgCl pellet electrode embedded in a silver wire outside of the QNM.</li>
	<li>Once the test cell is set up, turn on the power supply and data acquisition program. Ensure that the DC current reading is 0 pA in the absence of solution in the test cell.</li>
	<li>Use a syringe connected to the test cell via a fluid line to add buffered electrolyte solution above the face of the QNM and to ensure that the ionic current saturates the amplifier. If it does not, the QNM may be clogged. Apply a pop voltage (&#177; 1 V) and/or a pressure greater than 300 mm Hg to clear it. If that works, reduce the voltage and pressure.</li>
</ol>
3. Lipid Bilayer Formation
<ol>
	<li>Fill the solution in the test cell so that the solution level is well above the face of the QNM. Then lower the solution level via syringe to below the face, such that the current decreases to zero.</li>
	<li>Dip a 10 &#956;L pipette tip into the lipid vial. Push on the back end of the pipette tip and tap it on the side of the vial to remove all visible lipid.</li>
	<li>Touch the pipette tip onto the air-water interface of the solution in the test cell when the solution level is above the QNM&#8217;s face and wait two to five minutes for the lipid to spread uniformly.</li>
	<li>Slowly lower the solution level below the face of the QNM until the current saturates, and then slowly raise the solution level past the face of the QNM to form a lipid bilayer membrane.
	<ol>
		<li>Once a bilayer appears to form (i.e., when the current goes to zero), try popping it several times by increasing the pressure and ensure that the QNM is not clogged. To reform the lipid bilayer membrane, lower the solution level below the face of the QNM and slowly raise it.</li>
	</ol>
	</li>
	<li>If the lipid bilayer membrane did not form the first time, lower the solution below the face and raise it again. If it does not form after 3 trials, add more&#160;lipids&#160;as described in 3.2 and 3.3.</li>
	<li>After forming a membrane, set the current offset to zero when the applied potential is zero.</li>
</ol>
4. &#945;HL Pore Formation
<ol>
	<li>Add 2.5 ng of purified preformed&#160;&#945;HL heptameric protein sample (or 250 ng of monomeric &#945;HL) to the test cell (volume &#8776; 200 &#956;L) to enable channel formation.</li>
	<li>Increase the pressure on the bilayer with a gas-tight syringe (Figure 1) after a bilayer is formed, to expand the membrane from the QNM, and facilitate nanopore insertion. Raise the applied back pressure typically between 40 to 200 mmHg, depending on each QNM. 
	Note: The EBS software has an automated insertion feature that applies a higher bias (typically 200 to 400 mV) to induce pore formation and then automatically reduces the desired voltage to the measurement bias once a single channel forms.</li>
	<li>After a nanopore forms, reduce the back-pressure to about 1&#8260;2 of the insertion pressure. If multiple channels are observed, remove them by significantly reducing the pressure.</li>
</ol>
5. Metallic Cluster Partitioning in the Nanopore
<ol>
	<li>To account for electrode imbalances, set the DC offset voltage such that when the applied potential is set to zero there is no measured current.</li>
	<li>Prior to adding the POM sample, perform a control experiment to ensure there are no contaminants (e.g., trace POMs from a previous experiment) in the reservoir. Specifically, acquire an ionic current trace under an applied potential of +120 mV to -120 mV in the absence of any POMs to verify that no spontaneous current blockades are present. 
	Note: Due to the asymmetric structure of the &#945;HL channel (Figure 1), the magnitute of the ionic current will differ for positive and negative applied potentials. The ratio of the measured current above this applied voltage is indicative of the orientation of the &#945;HL nanopore in the membrane.</li>
	<li>Add the POM sample by flushing the reservoir with metallic cluster solution at 1 to 5 &#956;M concentration. Alternatively, load the sample into the capillary prior to cell assembly to study the partitioning of POMs into the other end of the &#945;HL channel.</li>
	<li>Record the ionic current using the manufacturer&#8217;s software to detect transient current blockades caused by partitioning of individual POMs into the nanopore. Estimate the physical and chemical properties of the molecule from the ionic current blockade depth, event frequency, and the residence time distribution of the blockades.</li>
</ol>
6. Ion Channel Recordings and Data Analysis
<ol>
	<li>Acquire the ionic current time series measurements using a high-impedance, low-noise amplifier and data acquisition system. Perform the measurements at an applied voltage of -120 mV (relative to channel cis side) for each pH.</li>
	<li>Apply a low-pass 100 kHz 8-pole Bessel filter to the signal, which is subsequently digitized at 500 kHz (i.e., 2 &#956;s/point). Extract events from the time series and analyze events using the ADEPT algorithm in the MOSAIC software package32,33.</li>
</ol>

<ol>
	<li>Ettedgui, J., Kasianowicz, J. J., Balijepalli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+discrimination+of+heteropolytungstates+and+their+Isomers+in+solution+with+a+nanometer-scale+pore.">Single molecule discrimination of heteropolytungstates and their Isomers in solution with a nanometer-scale pore.</a> Journal of the American Chemical Society. 138 (23), 7228-7231 (2016).</li><li>Bezrukov, S., Kasianowicz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+noise+reveals+protonation+kinetics+and+number+of+ionizable+sites+in+an+open+protein+ion+channel.">Current noise reveals protonation kinetics and number of ionizable sites in an open protein ion channel.</a> Physical Review Letters. 70 (15), 2352-2355 (1993).</li><li>Kasianowicz, J. J., Bezrukov, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protonation+dynamics+of+the+alpha-toxin+ion+channel+from+spectral+analysis+of+pH-dependent+current+fluctuations.">Protonation dynamics of the alpha-toxin ion channel from spectral analysis of pH-dependent current fluctuations.</a> Biophysj. 69 (1), 94-105 (1995).</li><li>Please, T. R., Ayub, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-State+Nanopore.">Solid-State Nanopore.</a> Engineered Nanopores for Bioanalytical Applications. , 121-140 (2013).</li><li>Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-state+nanopores.">Solid-state nanopores.</a> Nature Nanotechnology. 2 (4), 209-215 (2007).</li><li>Kasianowicz, J. J., Brandin, E., Branton, D., Deamer, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+individual+polynucleotide+molecules+using+a+membrane+channel.">Characterization of individual polynucleotide molecules using a membrane channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (24), 13770-13773 (1996).</li><li>Akeson, M., 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=Microsecond+time-scale+discrimination+among+polycytidylic+acid,+polyadenylic+acid,+and+polyuridylic+acid+as+homopolymers+or+as+segments+within+single+RNA+molecules.">Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules.</a> Biophysical Journal. 77 (6), 3227-3233 (1999).</li><li>Singer, A., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopore-based+Sensing+of+Individual+Nucleic+Acid+Complexes.">Nanopore-based Sensing of Individual Nucleic Acid Complexes.</a> Israel Journal of Chemistry. 49 (3-4), 323-331 (2010).</li><li>Jin, Q., Fleming, A. M., Burrows, C. J., White, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unzipping+kinetics+of+duplex+DNA+containing+oxidized+lesions+in+an+&#945;-hemolysin+nanopore.">Unzipping kinetics of duplex DNA containing oxidized lesions in an &#945;-hemolysin nanopore.</a> Journal of the American Chemical Society. 134 (26), 11006-11011 (2012).</li><li>Halverson, K. M., 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=Anthrax+biosensor,+protective+antigen+ion+channel+asymmetric+blockade.">Anthrax biosensor, protective antigen ion channel asymmetric blockade.</a> Journal of Biological Chemistry. 280 (40), 34056-34062 (2005).</li><li>Oukhaled, G., 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=Unfolding+of+proteins+and+long+transient+conformations+detected+by+single+nanopore+recording.">Unfolding of proteins and long transient conformations detected by single nanopore recording.</a> Physical Review Letters. 98 (15), 158101 (2007).</li><li>Reiner, J. E., Kasianowicz, J. J., Nablo, B. J., Robertson, J. W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+for+polymer+analysis+using+nanopore-based+single-molecule+mass+spectrometry.">Theory for polymer analysis using nanopore-based single-molecule mass spectrometry.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (27), 12080-12085 (2010).</li><li>Robertson, J. W. F., 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=Single-molecule+mass+spectrometry+in+solution+using+a+solitary+nanopore.">Single-molecule mass spectrometry in solution using a solitary nanopore.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (20), 8207-8211 (2007).</li><li>Baaken, G., Ankri, N., Schuler, A. -. K., R&#252;he, J., Behrends, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopore-based+single-molecule+mass+spectrometry+on+a+lipid+membrane+microarray.">Nanopore-based single-molecule mass spectrometry on a lipid membrane microarray.</a> ACS Nano. 5 (10), 8080-8088 (2011).</li><li>Angevine, C. E., Chavis, A. E., Kothalawala, N., Dass, A., Reiner, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+single+molecule+mass+spectrometry+via+charged+metallic+clusters.">Enhanced single molecule mass spectrometry via charged metallic clusters.</a> Analytical Chemistry. 86 (22), 11077-11085 (2014).</li><li>Astier, Y., Uzun, O., Stellacci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+study+of+single+gold+nanoparticle/alpha-Hemolysin+complex+formation:+a+nanotool+to+slow+down+ssDNA+through+the+alpha-Hemolysin+nanopore.">Electrophysiological study of single gold nanoparticle/alpha-Hemolysin complex formation: a nanotool to slow down ssDNA through the alpha-Hemolysin nanopore.</a> Small. 5 (11), 1273-1278 (2009).</li><li>Chavis, A. E., Brady, K. T., Kothalawala, N., Reiner, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+and+blockade+state+optimization+of+cluster-enhanced+nanopore+spectrometry.">Voltage and blockade state optimization of cluster-enhanced nanopore spectrometry.</a> Analyst. 140 (22), 7718-7725 (2015).</li><li>Campos, E., 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=Sensing+single+mixed-monolayer+protected+gold+nanoparticles+by+the+&#945;-hemolysin+nanopore.">Sensing single mixed-monolayer protected gold nanoparticles by the &#945;-hemolysin nanopore.</a> Analytical Chemistry. 85 (21), 10149-10158 (2013).</li><li>Campos, E., 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=The+role+of+Lys147+in+the+interaction+between+MPSA-gold+nanoparticles+and+the+&#945;-hemolysin+nanopore.">The role of Lys147 in the interaction between MPSA-gold nanoparticles and the &#945;-hemolysin nanopore.</a> Langmuir. 28 (44), 15643-15650 (2012).</li><li>Baaken, G., 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=High-Resolution+Size-Discrimination+of+Single+Nonionic+Synthetic+Polymers+with+a+Highly+Charged+Biological+Nanopore.">High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore.</a> ACS Nano. 9 (6), 6443-6449 (2015).</li><li>Berzelius, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beitrag+zur+n&#228;heren+Kenntniss+des+Molybd&#228;ns.">Beitrag zur n&#228;heren Kenntniss des Molybd&#228;ns.</a> Annalen Der Physik. 82 (1), 369-392 (1826).</li><li>Long, D. -. L., Burkholder, E., Cronin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyoxometalate+clusters,+nanostructures+and+materials:+from+self+assembly+to+designer+materials+and+devices.">Polyoxometalate clusters, nanostructures and materials: from self assembly to designer materials and devices.</a> Chemical Society Reviews. 36 (1), 105-121 (2007).</li><li>Muller, A., 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=Polyoxovanadates:+High-nuclearity+spin+clusters+with+interesting+host-guest+systems+and+different+electron+populations.+Synthesis,+spin+organization,+magnetochemistry,+and+spectroscopic+studies.">Polyoxovanadates: High-nuclearity spin clusters with interesting host-guest systems and different electron populations. Synthesis, spin organization, magnetochemistry, and spectroscopic studies.</a> Inorganic Chemistry. 36 (23), 5239-5250 (1997).</li><li>Rausch, B., Symes, M. D., Chisholm, G., Cronin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoupled+catalytic+hydrogen+evolution+from+a+molecular+metal+oxide+redox+mediator+in+water+splitting.">Decoupled catalytic hydrogen evolution from a molecular metal oxide redox mediator in water splitting.</a> Science. 345 (6202), 1326-1330 (2014).</li><li>Dolbecq, A., Dumas, E., Mayer, C. R., Mialane, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+organic-inorganic+polyoxometalate+compounds:+from+structural+diversity+to+applications.">Hybrid organic-inorganic polyoxometalate compounds: from structural diversity to applications.</a> Chemical Reviews. 110 (10), 6009-6048 (2010).</li><li>Busche, C., 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=Design+and+fabrication+of+memory+devices+based+on+nanoscale+polyoxometalate+clusters.">Design and fabrication of memory devices based on nanoscale polyoxometalate clusters.</a> Nature. 515 (7528), 545-549 (2014).</li><li>Pope, M., M&#252;ller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polyoxometalates: From Platonic Solids to Anti-Retroviral Activity. 10, (2012).</li><li>Rhule, J. 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Heteropoly and Isopoly Oxometalates. 8, (1983).</li><li>Braha, O., 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=Designed+protein+pores+as+components+for+biosensors.">Designed protein pores as components for biosensors.</a> Chemistry &amp; Biology. 4 (7), 497-505 (1997).</li><li>Forstater, J. H., 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=MOSAIC:+A+modular+single-molecule+analysis+interface+for+decoding+multistate+nanopore+data.">MOSAIC: A modular single-molecule analysis interface for decoding multistate nanopore data.</a> Analytical Chemistry. 88 (23), 11900-11907 (2016).</li><li>Balijepalli, A., 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=Quantifying+Short-Lived+Events+in+Multistate+Ionic+Current+Measurements.">Quantifying Short-Lived Events in Multistate Ionic Current Measurements.</a> ACS Nano. 8, 1547-1553 (2014).</li><li>Misakian, M. M., Kasianowicz, J. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrostatic+influence+on+ion+transport+through+the+alphaHL+channel.">Electrostatic influence on ion transport through the alphaHL channel.</a> Journal of Membrane Biology. 195 (3), 137-146 (2003).</li><li>Piguet, F., 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=Identification+of+single+amino+acid+differences+in+uniformly+charged+homopolymeric+peptides+with+aerolysin+nanopore.">Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore.</a> Nature Communication. 9 (966), (2018).</li></ol>

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>&lt;em&gt;Decane, ReagentPlus, &amp;ge;99%,&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>D901</td><td></td></tr><tr><td>&amp;alpha;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 ...

high-resolution-physical-characterization-single-metallic

Research

JoVE Journal

Chemistry

5.7K Views.  National Institute of Standards and Technology. 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.

Video: High Resolution Physical Characterization of Single Metallic Nanoparticles

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

Engineering

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.
Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of &gt; 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.
Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.

The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described.

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial ...

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

A Continuous-flow Photocatalytic Reactor for the Precisely Controlled Deposition of Metallic Nanoparticles

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic nanoparticles is developed. Guidelines for the replication of the reactor and its operation are provided in detail. Three different composite systems (Pt/graphene, Pt/TiO2, and Au/TiO2) with monodisperse and uniformly distributed particles are produced by this reactor, and the photodeposition mechanism, as well as the synthesis optimization strategy, are discussed. The synthesis methods and their technical aspects are described comprehensively. The role of the ultraviolet (UV) dose (in each excitation pulse) on the photodeposition process is investigated and the optimum values for each composite system are provided.

For a continuous and scalable synthesis of noble-metal-based nanocomposites, a novel photocatalytic reactor is developed and its structure, operation principles, and product quality optimization strategies are described.

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Bioengineering

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1&#8211;100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5&#8211;100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Synthesizing Nanoparticles and Nanostructures via Laser Ablation

Ultrafast Laser-Ablated Nanoparticles and Nanostructures for Surface-Enhanced Raman Scattering-Based Sensing Applications

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields such as sensing, catalysis, and medicine. The exceptional feature of this technique is the formation of nanoparticles (colloids) and nanostructures (solids) in a single experiment with ultrashort laser pulses. We have been working on this technique for the past few years, investigating its potential using the surface-enhanced Raman scattering (SERS) technique in hazardous materials sensing applications. Ultrafast laser-ablated substrates (solids and colloids) could detect several analyte molecules at the trace levels/mixture form, including dyes, explosives, pesticides, and biomolecules. Here, we present some of the results achieved using the targets of Ag, Au, Ag-Au, and Si. We have optimized the nanostructures (NSs) and nanoparticles (NPs) obtained (in liquids and air) using different pulse durations, wavelengths, energies, pulse shapes, and writing geometries. Thus, various NSs and NPs were tested for their efficiency in sensing numerous analyte molecules using a simple, portable Raman spectrometer. This methodology, once optimized, paves the way for on-field sensing applications. We discuss the protocols in (a) synthesizing the NPs/NSs via laser ablation, (b) characterization of NPs/NSs, and (c) their utilization in the SERS-based sensing studies.

Author Spotlight: Advancements and Applications in Nanoparticle Synthesis Through Laser Ablation in Liquids 

Ultrafast laser ablation in liquid is a precise and versatile technique for synthesizing nanomaterials (nanoparticles [NPs] and nanostructures [NSs]) in liquid/air environments. The laser-ablated nanomaterials can be functionalized with Raman-active molecules to enhance the Raman signal of analytes placed on or near the NSs/NPs.

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields ...