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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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 diameter of the nanopipette.
After fabrication of the nanopipette, a chemical reaction generates the solid nanotip inside the nanopipette. In this protocol, a gold nanotip is formed by the reduction of HAuCl4; other metal nanotips can be fabricated by designing corresponding reactions. After full blockage of the nanopipette tip, the electrochemical polarization of the generated gold nanotip promotes its further growth according to bipolar electrochemistry. An in-situ electro-optical characterization system is then constructed to achieve simultaneous recordings of the current traces and optical information during the growth process of the gold nanotip.
For characterization, calcium ion fluorescence detection helps verify whether the gap is generated between the inner wall of the pipette and the gold nanotip. For a well-defined closed-type WNE, the tip area should be invariably dark in the fluorescence image. Also, SEM can be used to characterize both the nanopipette and closed-type WNE. FIB may be employed to sculpt along the sidewall of the closed-type WNE to expose inner metal for the subsequent SEM imaging. Therefore, the length and inside structure of close-type WNE can be determined. Following its characterization, the well-prepared closed-type WNE is capable to be used for further applications.
This protocol for closed-type WNE paves a new path for electrochemical measurements of single nanoparticles with high reproducibility. However, there are still some challenges and limitations in this fabrication process. The first limitation involves the diameter of the nanopipette tip. Theoretically, when the tip diameter decreases to a single-molecule size, the current resolution can be dramatically enhanced. However, it is challenging to pull a nanopipette with a diameter under 30 nm with the existing pulling strategy.
The potential of this closed-type WNE protocol can be expanded to practical applications in nanosensing. By incorporating traditional nanoelectrodes with a scanning electrochemical microscope, the closed-type WNE can reveal dynamic electrochemical mapping for some special 2-D/3-D nanomaterials. Moreover, the plasmonic resonance scattering of the gold nanotip can be used to simultaneously detect the electron transfer process by both electrical readout and optical recording. By virtue of its geometrical properties, the closed-type WNE with a conical nanotip is suited to cellular analysis with low mechanical damage.

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

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 finally separates and forms two parts with nanoscale conical tips. The pulling parameters are provided in Table 1 for fabricating 30 nm nanopipettes in our lab. It should be noted that the parameters can vary from different laser pullers. Experimenters should adjust the parameters according to the laser power, temperature, and humidity. After fabrication, SEM characterization is required to verify the true diameter of the nanopipette.
Figure 2 shows the procedure of generating a gold nanotip inside the nanopipette tip after the pulling process. First, the AuCl4- inside the nanopipette is consistently reduced by BH4- to generate a gold nanotip until the opening of nanopipettes is fully blocked. Then, the bipolar electrochemistry promotes the further growth of the gold nanotip. We used an in situ characterization system to monitor the fabrication process of the closed-type WNE by simultaneous recording of the current response and dark-field images (Figure 3). As for the SEM characterization, Figure 4 shows top view SEM images of the bare nanopipette and closed-type WNE. After FIB splitting, a side view SEM image provides the morphology of the gold nanotip inside the closed-type WNE. In the single-nanoparticle collision experiments, the gold nanoparticles are added to the trans&#160;side of the WNE. The outstanding noise performance of this CNE uncovers the hidden signals with a high signal frequency (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59003/59003fig1.jpg" /> 
Figure 1: Fabrication of nanopipettes. The procedure for fabrication is as follows: step 1) install a microcapillary in a laser puller; step 2) heat the middle of the capillary with a CO2 laser and apply force at the ends of the capillary to pull it; and step 3) the capillary tapers down and separates into two symmetric nanopipettes in several seconds. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59003/59003fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59003/59003fig2.jpg" /> 
Figure 2: Fabrication of the closed-type WNE. Step 1) HAuCl4 and NaBH4 solutions are added to the cis and trans&#160;sides of the nanopipette, respectively. AuCl4- is reduced by BH4- to generate gold at the nanopipette orifice. Step 2) After the orifice is blocked by generated gold, the bipolar electrochemical reaction takes place with the applied potential for further growth of the gold nanotip. Step 3) A closed-type WNE is finally fabricated with a micrometer-length gold nanotip. This figure has been modified with permission from previous work25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59003/59003fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59003/59003fig3.jpg" /> 
Figure 3: Simultaneous electrical monitoring and scattering recording during closed-type WNE fabrication. (A) After addition of NaBH4 in the trans side of the nanopipette, the current drops immediately from 0 pA. Then, the current trace experiences a quick transition due to the generation of gold. After ~150 s, the current returns to 0 pA, demonstrating complete blockage of the nanopipette. (B) Dark-field images during WNE fabrication at corresponding timepoints of 0 s, 10 s, 100 s, and 150 s.&#160;This figure has been modified with permission from previous work25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59003/59003fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59003/59003fig4.jpg" /> 
Figure 4: SEM characterizations of the nanopipette and closed-type WNE. (A) Top view SEM image of a pulled nanopipette with a diameter of 30 nm. (B) Top view SEM image of a closed-type WNE with a diameter of 30 nm. (C) Side view SEM image of a closed-type WNE after FIB splitting from the tip to the back of the nanopipette. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59003/59003fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59003/59003fig5.jpg" /> 
Figure 5: Single nanoparticle collision detection with a closed-type WNE. (A) 30 nm gold nanoparticles are added to the trans side solution. A pair of Ag/AgCl electrodes are employed to apply a bias potential of 300 mV. Insert: a typical spike signal of a 30 nm gold nanoparticle collision. (B) A current trace without nanoparticles and after addition of 30 nm gold nanoparticles in the trans&#160;side of the closed-type WNE. This figure has been modified with permission from previous work25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59003/59003fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles at JoVE.com

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

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 ...

a-closed-type-wireless-nanopore-electrode-for-analyzing-single

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.4K Views.  East China University of Science and Technology. Elektrochemisch meten van het intrinsieke kenmerk van enkel nanodeeltje is van groot belang in de nanowetenschap. Deze methode toont een eenvoudige maar zeer reproduceerbare manier om een draadloze nanoporielelektrode te bouwen voor snelle analyse van enkel nanodeeltjes. De grootte van de nanoelectrode kan oplopen tot 30 nanometer door deze eenvoudige fabricagemethode.De huidige resolutie en de temporele resolutie tijdens de analyse zijn respectievelijk 0,6 picoamps en 0,01 millisecond...

Video: A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

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

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3 and size control is nontrivial4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.

A methodology for preparing solid-state nanopores in solution for biomolecular translocation experiments is presented. By applying short pulses of high electric fields, the nanopore diameter can be controllably enlarged with subnanometer precision and its electrical noise characteristics significantly improved. This procedure is performed in situ using standard laboratory equipment under experimental conditions.

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the ...

Engineering

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

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 constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Bioengineering

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

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.

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

Environment

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

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

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 in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Genetics

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

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

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.

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

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;

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 ...

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

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

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.

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

﻿Analyzing the Heterogeneous Performance of Single Nanoparticles with Electrochemical Methods

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

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This nanoscale heterogeneity remains hidden during the ensemble-averaged characterization of nanoparticles. Electrochemical techniques have been developed to measure currents from single nanoparticles but do not provide information about the structure and identity of the molecules that undergo reactions at the electrode surface. Optical techniques such as surface-enhanced Raman scattering (SERS) microscopy and spectroscopy can detect electrochemical events on individual nanoparticles while simultaneously providing information on the vibrational modes of electrode surface species. In this paper, a protocol to track the electrochemical oxidation-reduction of Nile Blue (NB) on single Ag nanoparticles using SERS microscopy and spectroscopy is demonstrated. First, a detailed protocol for fabricating Ag nanoparticles on a smooth and semi-transparent Ag film is described. A dipolar plasmon mode aligned along the optical axis is formed between a single Ag nanoparticle and Ag film. The SERS emission from NB fixed between the nanoparticle and the film is coupled into the plasmon mode, and the high-angle emission is collected by a microscope objective to form a donut-shaped emission pattern. These donut-shaped SERS emission patterns allow for the unambiguous identification of single nanoparticles on the substrate, from which the SERS spectra can be collected. In this work, a method for employing the SERS substrate as a working electrode in an electrochemical cell compatible with an inverted optical microscope is provided. Finally, tracking the electrochemical oxidation-reduction of NB molecules on an individual Ag nanoparticle is shown. The setup and the protocol described here can be modified to study various electrochemical reactions on individual nanoparticles.

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

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

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This ...

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes can alter protein biophysics and reduce the usefulness of the data acquired. Plasmonic nanotweezers is a technique that uses localized surface plasmon resonance (LSPR) on gold nanostructures to enhance the electric field within a confined hotspot region. This field enhancement allows for the use of low laser powers to trap single nanoparticles far smaller than conventional optical tweezers, down to only a few nanometers in diameter, such as single proteins. Trapping of single protein molecules within the hotspot region induces a shift in the local refractive index (nprotein &#62; nwater), altering light scattering as a product of the molecule&#39;s polarisability, which is affected by its volume, shape anisotropy, and refractive index. An avalanche photodiode (APD) collects the subsequent changes in light scattering. These alterations can then be analyzed to determine changes in the trapped molecule, including its size, global conformation, and dynamics of conformational change over time. The incorporation of microfluidics within the system allows for controlled environmental changes and real-time monitoring of their subsequent effects on the molecule. In this protocol, we demonstrate the steps to trap single protein molecules, alter their environmental solution conditions, and monitor their corresponding conformational changes using a plasmonic nanotweezers system.

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.

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...

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