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 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.
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 ± 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.
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.
2. Preparation of Experimental Setup
3. Fabrication of Closed-Type WNE
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 side of the WNE. The outstanding noise performance of this CNE uncovers the hidden signals with a high signal frequency (Figure 5).
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. Please click here to view a larger version of this figure.
Figure 2: Fabrication of the closed-type WNE. Step 1) HAuCl4 and NaBH4 solutions are added to the cis and trans 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. Please click here to view a larger version of this figure.
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. This figure has been modified with permission from previous work25. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 side of the closed-type WNE. This figure has been modified with permission from previous work25. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
This research was supported by the National Natural Science Foundation of China (61871183,21834001), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023), the “Chen Guang” Project from the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (17CG27).
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 |