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 chlorid...

<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 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>HAuCl4</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 &#181;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 &#181;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 &#181;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>NaBH4</td>
		</tr>
		<tr>
			<td>Ti-U inverted dark-field microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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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...

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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 ...

Electrochemically measuring the intrinsic feature of single nanoparticle is of great importance in nanoscience. This method demonstrate a simple but highly reproducible way to build a wireless nanopore electrode for rapid single nanoparticle analysis. The size of the nanoelectrode can reach as much as 30 nanometers by this simple fabrication method.The current resolution and the temporal resolution during the analysis are 0.6 picoamps and 0.01 milliseconds, respectively. It is anticipated that this wireless nanopore electrode will be utilized for in vivo and non-invasive secular analysis because of the nanoscale size of the nanoelectrode tip. The localized surface plasmonic resonance property of the gold nano tip and the perfect optical vision of the cross nanopipettes could enable electric optical detection at nano-scale.Researchers interested in the fabrication of wireless nanopore electrode should master the nanopipette pouring process. Because this is a crucial step in the procedure. They should pay attention to the environmental temperature and the humidity when pouring the pipette.First add 4.8 milliliters of chloroauric acid with a mass fraction of 1%to 40 milliliters of deionized water with vigorous stirring. Then heat the solution to a boil. Quickly add 10 milliliters of a trisodium citrate solution with a mass fraction of 1%into the solution.Heat the solution for an additional 15 minutes until the final solution is red in color. Place quartz capillaries in a 15 milliliter centrifuge tube filled with acetone and clean in an ultrasonic cleaner for ten minutes. When finished, remove the acetone and add ethanol.Then place the centrifuge tube in the ultrasonic cleaner for an additional ten minutes of cleaning. Next, place the quartz capillaries into another 15 milliliter centrifuge tube with deionized water for removal of the ethanol, and perform ultrasonic cleaning for 10 minutes. Dry the quartz capillaries using a nitrogen gas stream and store them in a clean centrifuge tube.Following this, turn on a carbon dioxide laser puller and pre-heat for 15-20 minutes to ensure a steady laser power. Install a clean quartz capillary in the pre-heated carbon dioxide laser puller. Set the pulling parameters of heat, filament, velocity, delay, and pulling force on the panel of the carbon dioxide laser puller for a specific diameter.Fix the prepared nanopipette on a petri dish with a reusable adhesive for further characterization. Inject 10 microliters of the prepared chloroauric acid solution into the nanopipette with a microloader. Centrifuge the nanopipette for five minutes at around 1, 878 times G for the removal of air bubbles in the nanopipette.Following centrifugation, fix the nanopipette on a cover slip with previously prepared silicone rubber, and define the area inside the nanopipette as the cis side and outside as the trans side. After waiting five minutes for the rubber to cure, place the integrated ensemble on the objective table of an inverted microscope. Turn on and adjust the dark field illumination to focus the nanopipette tip under a 10x microscope objective.Change to a 40x objective for higher spacial resolution. Next place one silver silver chloride electrode inside the nanopipette. Then place a second grounded silver silver chloride electrode on the trans side.Connect the silver silver chloride electrodes to a pre amplifier. Turn on the current measurement system and the corresponding software for ionic current recording. Then set the applied potential to 300 millivolts.Now slowly add 150 microliters of sodium borohydride solution to the trans side to trigger the reaction between the chloroauric acid and the sodium borohydride. Simultaneously, electrically and optically record the current trace and the dark field image scattering spectra using the current measurement and dark field detection systems. Turn off the applied potential after the ionic current tracing returns to zero picoamps.Wash the prepared closed type WNE with flowing deionized water from the bottom to the tip. Change the solution in the trans and cis sides to a potassium chloride solution after fabrication of the closed type WNE. Transfer 50 microliters of the 30 nanomolar gold nanopartical solution to the trans side.Then record the current signal of single nanoparticle collision events at a potential of 300 millivolts. Finally, change the applied voltage to monitor the frequency, amplitude, and shape change of the current signal. The fabrication of a nanopipette includes three main steps.A microcapillary with an inner diameter of 0.5 millimeter and and outer diameter of 1 milliliter is fixed in the puller, and a laser is then 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 procedure of generating a gold nanotip inside the nanopipette tip, after the pulling process is shown here.An in situ characterization system was used to monitor the fabrication process of the closed type WNE by simultaneous recording of the current response and dark field images. Top view SEM images of the bare nanopipette and closed type WNE are shown here after focus ions beam 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 side of the WNE.The outstanding noise performance of this CNE uncovers the hidden signals with a high signal frequency. When generating the gold nano tip, a low applied potential should be used to generate the electrochemical interface. A high applied potential could speed up the generation of gold and result in defective structures in the nano tip.The high contrast resolution and the high special resolution of this method can help researchers further understand the electron transfer process at the nano scale. Sodium borohydride is dangerous and will react violently with water. Please be careful when preparing the Sodium borohydride solution.

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7.3K visualizações.  East China University of Science and Technology. Medir eletroquimicamente a característica intrínseca da nanopartícula única é de grande importância na nanociência. Este método demonstra uma maneira simples, mas altamente reprodutível de construir um eletrodo de nanoporos sem fio para análise rápida de nanopartículas únicas. O tamanho da nanoeletrídico pode chegar a até 30 nanômetros por este método de fabricação simples.A resolução atual e a resolução temporal durante a análise são 0,6 picoamps e 0,01 miliss...