A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Summary

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

Abstract

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.

Introduction

Nanoparticles have attracted tremendous attention due to diverse features such as their catalytic ability, particular optical features, electroactivity, and high surface-to-volume ratios^1,2,3,4. 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 responses^5,6,7. One of these approaches involves immobilizing or capturing an individual nanoparticle on the interface of the nanoelectrode for the study of electrocatalysis^8,9. 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 undisciplinable^10,11,12,13. 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 process^14,15. 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 analysis^{16,17,18,19,20}. Owing to its controllable fabrication, the nanopipette provides a nanoscale confinement, with a uniform diameter ranging from 30-200 nm by a laser capillary puller^21,22,23,24. 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 interface^25,26,27,28. 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 ± 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.

Protocol

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 MΩ cm at 25 °C). The prepared solutions were filtered using a 0.22 μm pore-size filter.

1. Preparation of KCl solution
   1. Dissolve 0.074 g of potassium chloride in 100 mL of deionized water.
2. Preparation of NaBH₄ solution
   1. Dissolve 0.018 g of sodium borohydride in 10 mL of ethanol.
3. Preparation of HAuCl₄ solution
   1. Dissolve 0.010 g of potassium chloride in 1 mL of 1% chloroauric acid solution.
4. Preparation of silicone rubber
   1. Mix silicone rubber containing Part A and Part B (see Table of Materials) at a ratio of 1:1 by volume.
   2. Use the mixed silicone rubber to paint a reaction area at the slide immediately during the pot time of 1 min.
   3. Cure the prepared silicone rubber on the slide for 5 min.
5. Preparation of gold nanoparticles³⁰
   1. Add 4.8 mL of chloroauric acid with a mass fraction of 1% into 40 mL of deionized water with vigorous stirring.
   2. Heat the solution to a boil.
   3. Quickly add 10 mL of a trisodium citrate solution with a mass fraction of 1% into the solution.
   4. 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.

2. Preparation of Experimental Setup

1. Preparation of current measurement system
   1. Turn on the current measurement system containing the current amplifier (see Table of Materials) and low-noise data acquisition system (see Table of Materials)
   2. Switch on the voltage-clamp mode.
   3. Set the filter bandwidth to 10 kHz and sampling rate to 100 kHz.
   4. 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).
   5. Ground the shell of the Faraday cage, shells of the amplifier, and inverted microscope system.
2. Setup of the dark-field detection system
   1. 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) = 0.8–0.95)] 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 450–490 nm.

3. Fabrication of Closed-Type WNE

1. Fabrication of nanopipettes
   1. Put the quartz capillaries (see Table of Materials) in a 15 mL centrifuge tube filled with acetone for 10 min of ultrasonic cleaning.
   2. Pour off acetone, then add ethanol into the same centrifuge tube.
   3. Put the centrifuge tube in an ultrasonic cleaner for 10 min of cleaning.
   4. Put the capillaries into another 15 mL centrifuge tube with deionized water for removal of the ethanol, with 10 min of ultrasonic cleaning.
   5. Continually ultrasonic clean the capillaries three times with deionized water to remove the residual ethanol.
   6. Dry the capillaries using a nitrogen gas stream.
   7. Keep the capillaries in a new, clean centrifuge tube.
   8. Turn on the CO₂ laser puller (see Table of Materials)
   9. Preheat the puller for 15-20 min to ensure a steady laser power.
   10. Install the cleaned capillary in the puller.
   11. 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).
   12. Fix the prepared nanopipette on a Petri dish with the reusable adhesive (see Table of Materials) for further characterization.
2. Fabrication of closed-type WNE
   1. Inject 10 μL of the prepared HAuCl₄ solution into the nanopipette with a microloader.
   2. 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.
   3. Fix the nanopipette on a coverslip with the prepared silicone rubber (see step 1.4) and define the area inside the nanopipette as the “cis” side and outside as the “trans” side.
   4. Wait 5 min until the rubber is cured.
   5. Put the integrated ensemble on the objective table of the inverted microscope.
   6. Turn on and adjust the dark-field illumination to focus the nanopipette tip under a 10X microscope objective.
   7. Change to 20X and 40X objectives for a higher spatial resolution.
   8. Place one Ag/AgCl electrode inside the nanopipette.
   9. Place the other grounded Ag/AgCl electrode into the trans side.
   10. Connect a pair of Ag/AgCl electrodes to the pre-amplifier.
   11. Turn on the current measurement system and the corresponding software (see Table of Materials) for ionic current recording.
   12. Set the applied potential to 300 mV.
   13. Slowly add 150 μL 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.
   14. 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.
   15. Turn off the applied potential after ionic current tracing back to 0 pA.
   16. Wash the prepared closed-type WNE with flowing deionized water from the bottom to the tip.
3. Characterization of closed-type WNE
   1. Characterize closed-type WNE with a scanning electron microscope (SEM), which is a general method for characterization of nanopipettes^{22,31,32,33,34}.
   2. Use a calcium ion fluorescent experiment to verify the sealing condition of the gold nanotip inside the nanopipette.
      1. Inject 10 μL of CaCl₂ solution into the cis side of the closed-type WNE and Fluo-8 solution into the trans side.
      2. Connect the Ag/AgCl electrodes to the headstage.
   3. 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.
4. Single nanoparticle collision with closed-type WNE
   1. Change the solution in the trans and cis sides to a KCl solution after fabrication of the closed-type WNE.
   2. Transfer 50 μL 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).
   3. Change the applied voltage to monitor the frequency, amplitude, and shape change of the current signal.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Acetone	Sigma-Aldrich	650501	Highly flammable and volatile
Analytical balance	Mettler Toledo	ME104E
Axopatch 200B amplifier	Molecular Devices
Blu-Tack reusable adhesive	Bostik
Centrifuge tube	Corning Inc.		Centrifuge Tubes with CentriStar Cap, 15 ml
Chloroauric acid	Energy Chemical	E0601760010	HAuCl4
Clampfit 10.4 software	Molecular Devices
Digidata 1550A digitizer	Molecular Devices
DS Fi1c true-color CCD camera	Nikon
Ecoflex 5 Addtion cure silicone rubber	Smooth-On	17050377
Eppendorf Reference 2 pipettes	Eppendorf	492000904	10, 100 and 1000 µL
Ethanol	Sigma-Aldrich	24102	Highly flammable and volatile
Faraday cage			Copper
iXon 888 EMCCD	Andor
Microcentrifuge tubes	Axygen Scientific		0.6, 1.5 and 2.0 mL
Microloader	Eppendorf	5242 956.003	20 µL
Microscope Cover Glass	Fisher Scientific	LOT 16938	20 mm*60 mm-1 mm thick
Milli-Q water purifier	Millipore	SIMS00000	Denton Electron Beam Evaporator
P-2000 laser puller	Sutter Instrument
Pipette tips	Axygen Scientific		10, 200 and 1,000 µL
Potassium chloride,+D25+A2:F2+A2:F25	Sigma Aldrich	P9333-500G	KCl
Quartz pipettes	Sutter	QF100-50-7.5	O.D.:1.0 mm, I.D.:0.5 mm, 75 mm length
Refrigerator	Siemens
Silicone thinner	Smooth-On	1506330
Silver wire	Alfa Aesar	11466
Sodium borohydride,	Tianlian Chem. Tech.	71320	NaBH4
Ti-U inverted dark-field microscope	Nikon