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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 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 α-hemolysin (αHL), 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 nanoparticles^{15,16,17,18,19}, and other synthetic molecules²⁰.

We recently demonstrated that the αHL 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 chemistry^22,23, catalysis²⁴, material science^25,26, and biomedical research^27,28,29.

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, XM₁₂O₄₀^q- 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.

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 Analyte Preparation Prepare all electrolyte solutions with 18 MΩ-cm water from a Type-1 water purification system to remove trace organic species and then filter all electrolyte solutions through a 0.22 μm 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. Wild Type αHL. Follow MSDS precautions when handling the αHL toxin protein. Mix lyophilized wild-type monomeric S. aureus α-Hemolysin (αHL) powder with 18 MΩ-cm water at 1 mg/mL. Distribute 10 to 30 μL aliquots of the sample into cryo-safe centrifuge tubes, quickly flash freeze in liquid nitrogen and then store at -80 °C. Alternatively, use purified preformed heptameric αHL31. 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 °C for repeated use for up to one month. Prepare phosphotungstic acid solutions. Follow MSDS precautions when handling the phosphotungstic acid 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. 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-. 2. Test Cell Assembly Assemble the test cell per the manufacturer’s instructions. 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). Place a cylindrical AgCl pellet electrode embedded in a silver wire outside of the QNM. 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. 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 (± 1 V) and/or a pressure greater than 300 mm Hg to clear it. If that works, reduce the voltage and pressure. 3. Lipid Bilayer Formation 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. Dip a 10 μL 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. Touch the pipette tip onto the air-water interface of the solution in the test cell when the solution level is above the QNM’s face and wait two to five minutes for the lipid to spread uniformly. 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. 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. 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 lipids as described in 3.2 and 3.3. After forming a membrane, set the current offset to zero when the applied potential is zero. 4. αHL Pore Formation Add 2.5 ng of purified preformed αHL heptameric protein sample (or 250 ng of monomeric αHL) to the test cell (volume ≈ 200 μL) to enable channel formation. 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. After a nanopore forms, reduce the back-pressure to about 1⁄2 of the insertion pressure. If multiple channels are observed, remove them by significantly reducing the pressure. 5. Metallic Cluster Partitioning in the Nanopore 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. 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 αHL 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 αHL nanopore in the membrane. Add the POM sample by flushing the reservoir with metallic cluster solution at 1 to 5 μM concentration. Alternatively, load the sample into the capillary prior to cell assembly to study the partitioning of POMs into the other end of the αHL channel. Record the ionic current using the manufacturer’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. 6. Ion Channel Recordings and Data Analysis 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. Apply a low-pass 100 kHz 8-pole Bessel filter to the signal, which is subsequently digitized at 500 kHz (i.e., 2 μs/point). Extract events from the time series and analyze events using the ADEPT algorithm in the MOSAIC software package32,33.

Representative 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 im…

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

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
<em>Decane, ReagentPlus, &ge;99%,</em>	Sigma-Aldrich	D901
&alpha;HL	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