Name: A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles (Video) | JoVE Journal
Uploaded: 2019-03-20T13:00:00.000+00:00
Duration: 8 min 31 s
Description: Learn to fabricate a closed-type wireless nanopore electrode for rapid single nanoparticle analysis using electrochemical measurement. This method achieves 0.6 picoamp current resolution and 0.01 millisecond temporal resolution.

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5 minutes

This research was supported by the National Natural Science Foundation of China (61871183&#65292;21834001), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023), the &#8220;Chen Guang&#8221; Project from the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (17CG27).

1. Preparation of Solutions
NOTE: Pay attention to general safety precautions for all chemicals. Dispose of chemicals in a fume hood, and wear gloves, goggles, and a lab coat. Keep flammable liquids away from fire or sparks. All aqueous solutions were prepared using ultrapure water (18.2 M&#937; cm at 25 &#176;C). The prepared solutions were filtered using a 0.22 &#956;m pore-size filter.
<ol>
	<li>Preparation of KCl solution
	<ol>
		<li>Dissolve 0.074 g of potassium chloride in 100 mL of deionized water.</li>
	</ol>
	</li>
	<li>Preparation of NaBH4 solution 
	<ol>
		<li>Dissolve 0.018 g of sodium borohydride in 10 mL of ethanol.</li>
	</ol>
	</li>
	<li>Preparation of HAuCl4 solution
	<ol>
		<li>Dissolve 0.010 g of potassium chloride in 1 mL of 1% chloroauric acid solution.</li>
	</ol>
	</li>
	<li>Preparation of silicone rubber
	<ol>
		<li>Mix silicone rubber containing Part A and Part B (see Table of Materials) at a ratio of 1:1 by volume.</li>
		<li>Use the mixed silicone rubber to paint a reaction area at the slide immediately during the pot time of 1 min.</li>
		<li>Cure the prepared silicone rubber on the slide for 5 min.</li>
	</ol>
	</li>
	<li>Preparation of gold nanoparticles30
	<ol>
		<li>Add 4.8 mL of chloroauric acid with a mass fraction of 1% into 40 mL of deionized water with vigorous stirring.</li>
		<li>Heat the solution to a boil.</li>
		<li>Quickly add 10 mL of a trisodium citrate solution with a mass fraction of 1% into the solution.</li>
		<li>Heat the solution for an additional 15 min until the final solution is red in color. 
		NOTE: In our case, the chloroauric acid solution was rapidly reduced by trisodium citrate, and it was observed that the solution rapidly changed from clear-yellow to dark-black.</li>
	</ol>
	</li>
</ol>
2. Preparation of Experimental Setup
<ol>
	<li>Preparation of current measurement system
	<ol>
		<li>Turn on the current measurement system containing the current amplifier (see Table of Materials) and low-noise data acquisition system (see Table of Materials)</li>
		<li>Switch on the voltage-clamp mode.</li>
		<li>Set the filter bandwidth to 10 kHz and sampling rate to 100 kHz.</li>
		<li>Assemble a self-designed specific homemade copper cage to shield external noise for experimental cells and the pre-amplifier on the inverted microscope (see Table of Materials).</li>
		<li>Ground the shell of the Faraday cage, shells of the amplifier, and inverted microscope system.</li>
	</ol>
	</li>
	<li>Setup of the dark-field detection system 
	<ol>
		<li>Generation of the gold nanotip inside the nanopipette is monitored by the dark-field microscope. 
		NOTE: An inverted microscope system (see Table of Materials) is used to take images and scatter spectra. A true-color digital CCD camera is employed to take images of the nanopipette and nanopore electrode. A dark-field condenser [numerical aperture (NA) = 0.8&#8211;0.95)] is utilized to form a dark-field illumination. 10X (NA = 0.3), 20X (NA = 0.45), and 40X (NA = 0.6) objectives are used to collect images of the closed-type WNE. Fluorescence detection is used to further verify whether there are gaps between the nanotip and inner wall of the nanopipette. This experiment is performed by another EMCCD (see Table of Materials) also integrated on the inverted microscope, and the excitation light is a built-in mercury lamp with a band-pass filter of 450&#8211;490 nm.</li>
	</ol>
	</li>
</ol>
3. Fabrication of Closed-Type WNE
<ol>
	<li>Fabrication of nanopipettes
	<ol>
		<li>Put the quartz capillaries (see Table of Materials) in a 15 mL centrifuge tube filled with acetone for 10 min of ultrasonic cleaning.</li>
		<li>Pour off&#160;acetone, then add ethanol into the same centrifuge tube.</li>
		<li>Put the centrifuge tube in an ultrasonic cleaner for 10 min of cleaning.</li>
		<li>Put the capillaries into another 15 mL centrifuge tube with deionized water for removal of the ethanol, with 10 min of ultrasonic cleaning.</li>
		<li>Continually ultrasonic clean the capillaries three times with deionized water to remove the residual ethanol.</li>
		<li>Dry the capillaries using a nitrogen gas stream.</li>
		<li>Keep the capillaries in a new, clean centrifuge tube.</li>
		<li>Turn on the CO2 laser puller (see Table of Materials)</li>
		<li>Preheat the puller for 15-20 min to ensure a steady laser power.</li>
		<li>Install the cleaned capillary in the puller.</li>
		<li>Set the pulling parameters of heat, filament, velocity, delay, and pulling force on the panel of the CO2 laser puller for a specific diameter. The detailed parameter for pulling a 30 nm diameter nanopipette in this protocol is shown in Table 1 (Figure 1).</li>
		<li>Fix the prepared nanopipette on a Petri dish with the reusable adhesive (see Table of Materials) for further characterization.</li>
	</ol>
	</li>
	<li>Fabrication of closed-type WNE
	<ol>
		<li>Inject 10 &#956;L of the prepared HAuCl4 solution into the nanopipette with a microloader.</li>
		<li>Centrifuge the nanopipette for 5 min at around 1878 x g for the removal of air bubbles in the nanopipette. 
		NOTE: For this step, we placed the nanopipette with the tip facing down into a homemade holder within a 2 mL centrifuge tube.</li>
		<li>Fix the nanopipette on a coverslip with the prepared silicone rubber (see step 1.4) and define the area inside the nanopipette as the &#8220;cis&#8221; side and outside as the &#8220;trans&#8221; side.</li>
		<li>Wait 5 min until the rubber is cured.</li>
		<li>Put the integrated ensemble on the objective table of the inverted microscope.</li>
		<li>Turn on and adjust the dark-field illumination to focus the nanopipette tip under a 10X microscope objective.</li>
		<li>Change to 20X and 40X objectives for a higher spatial resolution.</li>
		<li>Place one Ag/AgCl electrode inside the nanopipette.</li>
		<li>Place the other grounded Ag/AgCl electrode into the trans side.</li>
		<li>Connect a pair of Ag/AgCl electrodes to the pre-amplifier.</li>
		<li>Turn on the current measurement system and the corresponding software (see Table of Materials) for ionic current recording.</li>
		<li>Set the applied potential to 300 mV.</li>
		<li>Slowly add 150 &#956;L of NaBH4 solution into the trans side to trigger the reaction between HAuCl4 and NaBH4 (Figure 2). 
		NOTE: The reduction of NaBH4 in aqueous solution takes place at a violent reaction rate. Therefore, the generation of H2 from the reduction of NaBH4 may induce a defective structure of the nanotip by the generation of cavities during the gold nanotip growth.</li>
		<li>Simultaneously, electrically and optically record the current trace and the dark-field image/scattering spectra using the current measurement and dark-field detection systems (Figure 3). 
		NOTE: The ethanol solution is volatile under dark-field illumination. Pay attention to the volume of ethanol during the fabrication process.</li>
		<li>Turn off the applied potential after ionic current tracing back to 0 pA.</li>
		<li>Wash the prepared closed-type WNE with flowing deionized water from the bottom to the tip.</li>
	</ol>
	</li>
	<li>Characterization of closed-type WNE
	<ol>
		<li>Characterize closed-type WNE with a scanning electron microscope (SEM), which is a general method for characterization of nanopipettes22,31,32,33,34.</li>
		<li>Use a calcium ion fluorescent experiment to verify the sealing condition of the gold nanotip inside the nanopipette.
		<ol>
			<li>Inject 10 &#956;L of CaCl2 solution into the cis side of the closed-type WNE and Fluo-8 solution into the trans side.</li>
			<li>Connect the Ag/AgCl electrodes to the headstage.</li>
		</ol>
		</li>
		<li>Apply a 400 mV bias potential and use the EMCCD (see Table of Materials) to monitor the fluorescence response at the tip area. Use focus ions beam (FIB) to sculpt the closed-type WNE from the tip to the bottom, then determine the length of the interior metal layer or nanotip with SEM characterization.</li>
	</ol>
	</li>
	<li>Single nanoparticle collision with closed-type WNE
	<ol>
		<li>Change the solution in the trans and cis sides to a KCl solution after fabrication of the closed-type WNE.</li>
		<li>Transfer 50 &#956;L of 30 nm gold nanoparticle solution into the trans side. Record the current signal of single nanoparticle collision events at a potential of 300 mV (Figure 5).</li>
		<li>Change the applied voltage to monitor the frequency, amplitude, and shape change of the current signal.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors declare no conflicts of interests.

Fabrication of a well-defined nanopipette is the first step in the closed-type WNE fabrication process. By focusing a CO2 laser onto the center of the capillary, one capillary separates into two symmetrical nanopipettes with nanoscale conical tips. The diameter is easily controlled, ranging from 30-200 nm, by adjusting the parameters of the laser puller. It is noted that the parameters for pulling can vary for different pipette pullers. The environmental temperature and humidity can also influence the final di...

Nanoparticles have attracted tremendous attention due to diverse features such as their catalytic ability, particular optical features, electroactivity, and high surface-to-volume ratios1,2,3,4. Electrochemical analysis of single nanoparticles is a direct method for understanding the intrinsic chemical and electrochemical processes at the nanoscale level. To achieve highly sensitive measurements of single nanoparticles, two electrochemical approaches have been previously applied to read out nanoparticle information from current responses5,6,7. One of these approaches involves immobilizing or capturing an individual nanoparticle on the interface of the nanoelectrode for the study of electrocatalysis8,9. The other strategy is driven by single nanoparticle collision with the surface of an electrode, which generates a transient current fluctuation from the dynamic redox process.
Both of these methods require a nanoscale ultrasensitive sensing interface that matches the diameter of single nanoparticles. However, traditional fabrication of nanoelectrodes has mainly incorporated the micro-electromechanical systems (MEMS) or laser pulling techniques, which are tedious and undisciplinable10,11,12,13. For example, MEMS-based fabrication of nanoelectrodes is expensive and requires the use of a clean room, restricting the massive production and popularization of nanoelectrodes. On the other hand, laser pulling fabrication of nanoelectrodes relies heavily on experience of the operators during the sealing and pulling of a metal wire inside the capillary. If the metal wire is not well-sealed in the capillary, the gap between the inner wall of the nanopipette and wire can dramatically introduce excess background current noise and enlarge the electroactive sensing area. These drawbacks largely decrease the sensitivity of the nanoelectrode. On the other hand, the existence of a gap can enlarge the electrode area and reduce sensitivity of the nanoelectrode. As a consequence, it is hard to guarantee a reproducible performance due to the uncontrollable electrode morphologies in each fabrication process14,15. Therefore, a general fabrication method of nanoelectrodes with excellent reproducibility is urgently needed to facilitate electrochemical exploration of the intrinsic features of single nanoparticles.
Recently, the nanopore technique has been developed as an elegant and label-free approach for single molecule analysis16,17,18,19,20. Owing to its controllable fabrication, the nanopipette provides a nanoscale confinement, with a uniform diameter ranging from 30-200 nm by a laser capillary puller21,22,23,24. Moreover, this simple and reproducible fabrication procedure ensures the generalization of the nanopipette. Recently, we proposed a wireless nanopore electrode (WNE), which does not require the sealing of a metal wire inside the nanopipette. Through a facile and reproducible fabrication process, the WNE possesses a nanoscale metal deposition within the nanopipette to form an electroactive interface25,26,27,28. Since the WNE possesses a well-defined structure and uniform morphology of its confinements, it achieves high current resolution as well as low resistance-capacitance (RC) time constant for performing high temporal resolution. We previously reported two types of WNEs, open-type and closed-type, for realizing single entity analysis. The open-type WNE employs a nanometal layer deposited on the inner wall of a nanopipette, which converts the faradic current of a single entity to the ionic current response26. Usually, the diameter of an open-type WNE is around 100 nm. To further decrease the diameter of WNE, we presented the closed-type WNE, in which a solid metal nanotip fully occupies the nanopipette tip through a chemical-electrochemical approach. This method can rapidly generate a 30 nm gold nanotip inside a nanopore confinement. The well-defined interface at the tip area of a closed-type WNE ensures a high signal-to-noise ratio for electrochemical measurements of single nanoparticles. As a charged gold nanoparticle collides with the closed-type WNE, an ultrafast charging-discharging process at the tip interface induces a capacitive feedback response (CFR) in the ionic current trace. Compared to a previous single nanoparticle collision study via a nanoelectrode with metal wire inside29, the closed-type WNE showed a higher current resolution of 0.6 pA &#177; 0.1 pA (RMS) and higher temporal resolution of 0.01 ms.
Herein, we describe a detailed fabrication procedure for a closed-type WNE that has highly controlled dimensions and outstanding reproducibility. In this protocol, a simple reaction between AuCl4- and BH4- is designed to generate a gold nanotip that completely blocks the orifice of a nanopipette. Then, bipolar electrochemistry is adopted for continuous growth of a gold nanotip that reaches the length of several micrometers inside the nanopipette. This simple procedure enables the implementation of this nanoelectrode fabrication, which can be carried out in any general chemistry laboratory without a clean room and expensive equipment. To determine the size, morphology, and inner structure of a closed-type WNE, this protocol provides a detailed characterization procedure with use of a scanning electron microscope (SEM) and fluorescence spectroscopy. One recent example is highlighted, which directly measures the intrinsic and dynamic interactions of gold nanoparticles (AuNPs) colliding towards the nanointerface of a closed-type WNE. We believe that the closed-type WNE may pave a new path for future electrochemical studies of living cells, nanomaterials, and sensors at single-entity levels.

<table><tbody><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>650501</td><td>Highly flammable and volatile</td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>ME104E</td><td></td></tr><tr><td>Axopatch 200B amplifier</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Blu-Tack reusable adhesive</td><td>Bostik</td><td></td><td></td></tr><tr><td>Centrifuge tube</td><td>Corning Inc.</td><td></td><td>Centrifuge Tubes with CentriStar Cap, 15 ml</td></tr><tr><td>Chloroauric acid</td><td>Energy Chemical</td><td>E0601760010</td><td>HAuCl&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Clampfit 10.4 software</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Digidata 1550A digitizer</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>DS Fi1c true-color CCD camera</td><td>Nikon</td><td></td><td></td></tr><tr><td>Ecoflex 5 Addtion cure silicone rubber</td><td>Smooth-On</td><td>17050377</td><td></td></tr><tr><td>Eppendorf Reference 2 pipettes</td><td>Eppendorf</td><td>492000904</td><td>10, 100 and 1000 &amp;micro;L</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>24102</td><td>Highly flammable and volatile</td></tr><tr><td>Faraday cage</td><td></td><td></td><td>Copper</td></tr><tr><td>iXon 888 EMCCD</td><td>Andor</td><td></td><td></td></tr><tr><td>Microcentrifuge tubes</td><td>Axygen Scientific</td><td></td><td>0.6, 1.5 and 2.0 mL</td></tr><tr><td>Microloader</td><td>Eppendorf</td><td>5242 956.003</td><td>20 &amp;micro;L</td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific</td><td>LOT 16938</td><td>20 mm*60 mm-1 mm thick</td></tr><tr><td>Milli-Q water purifier</td><td>Millipore</td><td>SIMS00000</td><td>Denton Electron Beam Evaporator</td></tr><tr><td>P-2000 laser puller</td><td>Sutter Instrument</td><td></td><td></td></tr><tr><td>Pipette tips</td><td>Axygen Scientific</td><td></td><td>10, 200 and 1,000 &amp;micro;L</td></tr><tr><td>Potassium chloride,+D25+A2:F2+A2:F25</td><td>Sigma Aldrich</td><td>P9333-500G</td><td>KCl</td></tr><tr><td>Quartz pipettes</td><td>Sutter</td><td>QF100-50-7.5</td><td>O.D.:1.0 mm, I.D.:0.5 mm, 75 mm length</td></tr><tr><td>Refrigerator</td><td>Siemens</td><td></td><td></td></tr><tr><td>Silicone thinner</td><td>Smooth-On</td><td>1506330</td><td></td></tr><tr><td>Silver wire</td><td>Alfa Aesar</td><td>11466</td><td></td></tr><tr><td>Sodium borohydride,</td><td>Tianlian Chem. Tech.</td><td>71320</td><td>NaBH&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Ti-U inverted dark-field microscope</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

a closed-type wireless nanopore electrode for analyzing single nanoparticles

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in nanoscience. However, electrochemically analyzing single nanoparticles is challenging, as the sensing nanointerface is uncontrollable. To address this challenge, we describe here the fabrication and characterization of a closed-type wireless nanopore electrode (WNE) that exhibits a highly controllable morphology and outstanding reproducibility. The facile fabrication of WNE enables the preparation of well-defined nanoelectrodes in a general chemistry laboratory without the use of a clean room and expensive equipment. One application of a 30 nm closed-type WNE in analysis of single gold nanoparticles in the mixture is also highlighted, which shows a high current resolution of 0.6 pA and high temporal resolution of 0.01 ms. Accompanied by their excellent morphology and small diameters, more potential applications of closed-type WNEs can be expanded from nanoparticle characterization to single molecule/ion detection and single-cell probing.

We demonstrate a facile approach to fabricate a well-defined 30 nm wireless nanopore electrode based on a quartz conical nanopipette. The fabrication of a nanopipette is demonstrated in Figure 1, which includes three main steps. A microcapillary with an inner diameter of 0.5 mm and outer diameter of 1.0 mm is fixed in the puller, then a laser is focused on the center of the capillary to melt the quartz. By applying forces to the terminals of the capillary, it...

Here, we present a protocol for fabrication of a closed-type wireless nanopore electrode and subsequent electrochemical measurement of single nanoparticle collisions.

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in ...

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Research

JoVE Journal

Chemistry

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate...

Monitoring Protein Adsorption with Solid-state Nanopores

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Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

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Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

Nanopore DNA Sequencing for Metagenomic Soil Analysis

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.

High Resolution Physical Characterization of Single Metallic Nanoparticles

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

The protocol describes how to monitor electrochemical events on single nanoparticles using surface-enhanced Raman scattering spectroscopy and imaging.

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy

Plasmonic nanotweezers use localized surface plasmon resonance in gold nanostructures to trap single nanoparticles, including proteins, within a nanometer-scale optical field. Changes in the scattered signal reveal protein presence and conformational dynamics, enabling monitoring without fluorophore modifications or surface tethering.

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Learn to fabricate a closed-type wireless nanopore electrode for rapid single nanoparticle analysis using electrochemical measurement. This method achieves 0.6 picoamp current resolution and 0.01 millisecond temporal resolution.

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

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