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Facile Fabrication of Gold Functionalized Nanopipette for Nanoscale Electrochemistry and Surface Enhanced Raman Spectroscopy
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 47, Issue 8, August 2019 Online English edition of the Chinese language journal
Cite this article as: Chinese J. Anal. Chem., 2019, 47(8): e19104–e19112
RESEARCH PAPER
Facile Fabrication of Gold Functionalized Nanopipette for Nanoscale Electrochemistry and Surface Enhanced Raman Spectroscopy LI Hong-Na, YANG Dan, LIU Ao-Xue, LIU Guo-Hui, SHAN Yu-Ping, YANG Guo-Cheng*, HE Jin* School of Chemistry and Life Science, Advanced Institute of Materials Science, Changchun University of Technology, Changchun 130012, China
Abstract:
Nanopipette has emerged as a versatile nanosensor. It is desirable to expand the sensing capabilities of nanopipette by
integrating multiple sensing methods into one nanopipette. In this work, we presented a seeded growth approach to facilely, cheaply and quickly deposit a gold layer with controllable length at the inner surface of the nanopipette tip. The deposited gold formed a wireless ring-shape gold nanoelectrode at the nanopipette tip and enabled multimode single entity detection. With the presence of gold at the nanopipette apex, the nanopore based resistive-pulse sensing capability was obviously improved. The integrated ring-shape gold nanoelectrode could be used as a wireless bipolar electrode for electrochemical measurement. We further demonstrated that the gold coated nanopipette could be used as a substrate for surface enhanced Raman spectroscopy. Key Words:
Nanopipette; electrochemical method; Bipolar nanoelectrode; Raman spectroscopy
1 Introduction Nanopore technology provides a promising method to monitor the dynamics of single entity in real-time in ionic solutions. As a well-established single-entity nanosensor, it has been extensively used in applications such as DNA sequencing[1,2], single protein analysis[3,4] and single nanoparticle detection[5,6]. The basic nanopore sensing method is based on the resistive-pulse sensing[7]. A constant voltage is applied across the nanopore to drive the charged molecule or nanoparticle to move through a nanopore with a comparable size. The single-entity translocation event causes a transient ionic current change in the current-time trace. It is critical to fabricate size-controllable single nanopore for the analysis and detection of a ranges of nanoscale objects. Nanopipette is a special type of nanopore with a conical shape. Different from other solid-state nanopores, the fabrication of quartz/glass nanopipette with variable pore sizes is cheap, simple and reproducible. Nanopipettes have been developed
as effective chemical and biological sensors and exhibit a sensitivity at single-molecule level for DNA and protein studies[8,9]. Uniquely, the nanopipette can also be conveniently used for single living cell analysis[10,11]. It is of great interest to use nanopipette as a functional biosensor. However, it is still challenging for detection of analyte in a complicated environment, such as cytoplasm and extracellular fluid. To improve its sensitivity and selectivity, previous studies modified the nanopipette inner surface with carbon[12‒14], metal[15] or recognition molecules[16,17], while keeping the nanopore open. One approach is to fabricate metalized nanopipettes to integrate different sensing modalities, which can enhance both the sensitivity and selectivity of the nanopipette sensor. Various methods have been developed to fabricate nanoelectrodes, such as electron beam evaporation[18] to fabricate metallic nanoelectrode, chemical vapor deposition (CVD)[19] to fabricate carbon nanoelectrode, and laser pulling method[20] to fabricate a platinum or gold nanoelectrode. The conductive nanotip has
________________________ Received 18 March 2019; accepted 7 May 2019 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 21673023, 21773017). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61177-1
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
been investigated and reported by several groups [21,22]. For example, the work of nanoelectrode fabrication reported by Hao et al[23] suggested a new electrochemically plating approach by using liquid gallium/indium alloy to deposit gold on the nanopore of nanopipette. However, the fabrication process needs focused ion beam (FIB) milling method to expose the fresh metal nanoelectrode after gold deposition, which is time consuming and the yield is low. Xu et al[24] used a facile one-step photochemical approach to deposit an ultrathin gold film on the inner surface of the nanopipette tip. However, the deposition process is still very slow. In this work, we developed a gold nanoparticle (GNP) seed based electrochemical deposition method to deposit gold on the inner surface of the nanopipette tip. The gold deposition speed is fast, and the deposition process is self-terminated in a few minutes. After thermal annealing, ring-shape gold nanoelectrode and gold coated nanopore are formed at the nanopipette tip. By using GNPs as the model analyte, the gold coated nanopore shows improved signal-to-noise ratio (SNR) for the nanopore based resistive-pulse sensing method. In addition, the presence of gold at the nanopipette tip introduces new functions. The gold nanoelectrode is suitable for nanoscale electrochemical measurements and surface enhanced Raman spectroscopy (SERS). Besides, the ringshape gold at the nanopipette tip can be used as a bipolar nanoelectrode and as an effective substrate for SERS.
2
Experimental
2.1
Chemicals
Potassium (KCl) and sodium chloride (NaCl) were purchased from Beijing Chemical Works. Sulfuric acid (H2SO4), hydrogen peroxide (H2O2), sodium borohydride (NaBH4), and trisodium citrate (Na3C6H5O7∙2H2O) were purchased from Sinopharm Group. Gold chloride (HAuCl4∙xH2O) was purchased from Macklin. Hydroxylamine hydrochloride (NH2OH∙HCl) was purchased from Xilong Chemical Co., Ltd. Potassium ferricyanide (K3Fe(CN)6), ferrocenemethanol (FcMeOH), 4-aminothiophenol (4-ATP), monopotassium phosphate (KH2PO4), disodium phosphate (Na2HPO4) and sodium hypochlorite were purchased from Aladdin. The solutions were prepared using deionized (DI) water (18.2 MΩ cm at 25 ºC) from a water purification system (Millipore S.A.S). All reagents and materials were of at least analytical grade. The 50 nm silver nanoparticles (AgNPs) were prepared by reduction of silver nitrate with trisodium citrate using a well-established method[25]. 2.2
Fabrication of nanopipette
The
quartz
capillary
tubes
with
filament
(FG-G
QT100-50-7.5, Sutter Instrument) were used to fabricate the gold nanoelectrode. The pipettes were first cleaned by Piranha (the volume ratio of H2SO4 to H2O2 is 3:1, caution: Piranha solutions are highly corrosive and need to be handled with extreme caution.) for 30 min, then repeatedly rinsed with DI water and dried in an oven at 100 °C for overnight. The quartz nanopipettes with nanopore diameter of about 100 nm were fabricated from these cleaned capillary tubes by using a laser pipette puller (P-2000, Sutter Instrument) with following parameters 1: HEAT = 650, FIL = 4, VEL = 60, DEL = 145, PUL = 100. The quartz capillary tube is pulled with the following parameters 2: HEAT = 700, FIL = 3, VEL = 40, DEL = 175, PUL = 190 to fabricate the nanopore with about 45 nm diameter. 2.3
Measurements
Cyclic voltammogram (CV) and current-time (i-t) traces during gold deposition were acquired with a CHI852c electrochemical workstation. The Ag/AgCl wire electrode was used as the quasi-reference electrode in all electrochemical experiments. Platinum wire was used as the counter electrode for CV measurements. A scan rate of 50 mV s‒1 was used for all CV curves. The current-voltage (i-V) curves were measured by using a low noise source measure meter (2636B Keithley). For the resistive-pulse measurement of nanopipette, the i-t traces for nanoparticle translocation experiments were measured by using Synthesized Function Generatou (Model DS345) to prove voltage and the corresponding data were recorded by an Axon digidata 1550B. Two Ag/AgCl wire electrodes were used as the electrodes. The electrode in the PBS (10 mM, pH 7.4) bath solution was always grounded and the Vpore was applied at the Ag/AgCl electrode inside the nanopipette barrel. The traces were recorded at a 15 kHz sampling rate with a 30 Hz low-pass filter. All the measurements were performed at room temperature (25 °C). The scanning electron microscopy (SEM) images of nanopipettes were obtained by a field-emission SEM instrument (JOEL7610) and the samples were coated with a thin Pt layer before imaging. The optical microscope images were acquired by a Nikon Ti-U inverted microscope with a 40× objective lens. 2.4
Preparation of GNP
GNP seeds with diameter of about 3.5 nm were synthesized by using a strong reducing reagent (NaBH4) according to the literature method[26]. To synthesize 20-nm size GNPs for the control experiment, the AuCl4– ion was reduced by trisodium citrate[27]. The concentrations of 3.5-nm GNPs and 20-nm GNPs in the original solutions were 51 and 0.81 nM, respectively[28].
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
2.5
SERS measurement
The gold coated nanopipette was immersed in 4-ATP ethanol solution (50 mM) for 12 h and then rinsed copiously by DI water. In the next step, the 4-ATP modified gold coated nanopipette was immersed in 50-nm AgNP solution (0.225 nM) for 4 h. The Raman spectrum analysis was performed on a Horiba LabRam HR Evolution Raman spectrometer with 532-nm laser line and exposure time of 10 s.
3 Results and discussion 3.1
Fabrication of gold coated nanopipette
NaBH4 as a strong reductive reagent is widely used for the reduction of AuCl4– ion. However, this reaction occurs at a violent reaction rate and the generation of hydrogen gas can cause many defective cavities in the deposited gold [29,30]. To avoid this problem, we instead use a mild reductive agent NH2OH∙HCl to deposit gold on the tip of nanopipette. Wang et al[31] reported a gold deposition method based on a mild reducing reagent (NH2OH∙HCl). However, this gold deposition process is extremely slow, which will last three days. Different from previous methods, we added small GNPs with diameter about 3.5 nm in the DI water as the seeds. As we discussed later, the GNP seeds in the bath solution greatly speeded up the gold deposition process at the nanopipette tip. The setup is shown in Fig.1A. The nanopipette barrel was filled with a mixture solution of NH2OH∙HCl and HAuCl4 at concentration ratio of 1:2. The concentration of NH2OH∙HCl was 25 mM. The bath solution contained 51 nM 3.5 nm GNP seeds. We maintained a zero bias between two Ag/AgCl quasi reference electrodes and monitored the current change during gold deposition. The chemical reaction during gold deposition
process can be expressed as: 6NH2OH + 2HAuCl4 = 2Au (s) + 3N2 (g) + 8HCl + 6H2O This reaction mainly occurs at the nanopipette orifice, where the bath solution and the barrel solution are mixed. Figure 1B shows a typical i-t trace during gold deposition. Based on the current change, the gold deposition is divided into three stages. In the first stage, a negative current is quickly established right after the nanopipette tip is immersed in the bath solution to close the circuit. The negative ionic current is generated because of the concentration gradient and the negative surface charge of nanopipette, which driving the AuCl4– ions inside the nanopipette barrel to move out of the nanopipette apex. The gold deposition process, starting from the nanopipette apex, is accelerated by the GNP seeds, which serving as the nucleation centers[32]. In stage II, the negative current gradually rises, becomes positive and reaches a positive maximum. This is attributed to the gradual adsorption of cations, such as NH3+OH, to the negatively charged inner wall surface. The surface charge becomes positive and the positive surface potential drives a positive ionic current. The negatively charged GNP seeds in the bath solution can also be brought into the barrel under a positive potential. At stage III, the positive current falls continuously until reaches zero. The decrease of ionic current is induced by the shrinkage of pore size due to gold deposition. At zero current, the barrel at the nanopipette apex is fully clogged and the gold deposition is terminated automatically. The whole deposition progress generally only lasts a few minutes. The gold deposition at the tip can be clearly revealed in the optical microscope image, as shown in Fig.1C. The length of the deposited gold is about 7–8 μm. After electrochemical deposition, the nanopipette was cleaned and annealed in air at 300 °C for 4 h in a muffle furnace to improve the quality of deposited gold and the adhesion between the gold and the nanopipette inner surface.
Fig.1 (A) Experimental setup for gold deposition on inner surface of nanopipette tip. The bath solution is DI water with GNP seeds (51 nM); (B) Electrochemical i-t trace during the gold deposition at nanopipette tip. (C) Optical microscope image of the deposited gold at the tip of nanopipette
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
Fig.2
SEM images of nanopipette before (A) and (B) after gold deposition and annealing. (C) I-V curves of bare nanopipette (a), before (b) and after (c) annealing at 300 °C for 4 h. I-V curves are measured in PBS solution (10 mM)
To optimize and understand the self-terminated seed assisted gold deposition mechanism, different growth conditions were optimized. The gold deposition at different conditions is discussed later. Especially, the gold deposition is very sensitive to the external bias, which regulates the movement of ions through the nanopore and controls the reaction rate. 3.2
Characterization of bare nanopipette and gold coated nanopipette
SEM images of bare nanopipette and gold coated nanopipette are shown in Fig.2A and Fig.2B, respectively. Before gold deposition, the diameter of the bare nanopipette was about 100 nm. After annealing the deposited gold, the pore diameter of gold coated nanopipette was reduced to about 40 nm. The typical I-V curves measured in PBS solution (10 mM) are shown in Fig.2C. After gold deposition, the current was almost zero in the I-V curve (green curve), suggesting the pore was almost fully blocked. However, after annealing, the current through the nanopipette was partially recovered (red curve), which indicated the formation of a ring shape gold nanoelectrode at the nanopipette tip. These measurement results suggested that the seed growthbased electrochemical deposition approach was successful and a ring shape gold nanoelectrode was formed after annealing. 3.3
3.5 nm GNP seeds in the bath solution while fixing the concentration ratio of NH2OH∙HCl to HAuCl4 of nanopipette barrel solution at 1:2. The optical microscope images of gold coated nanopipette with and without GNP seeds in the bath solution are shown in Figs. 3D and 3E respectively. With the 3.5 nm GNP seeds in the bath solution, the gold deposition began from the orifice of the nanopipette and extends a length of 7–8 μm inside the barrel (Fig.3D). The GNP seeds were accumulated towards the nanopipette tip and worked as nucleation centers to trigger gold deposition right from the orifice, where the bath and barrel solutions mixed. In contrast, without GNP seeds in the bath solution, no significant gold deposition was observed at the orifice of the nanopipette. The length of gold layer was only about 1 μm at the tip (Fig.3E). Most of gold aggregates in 5-µm size were only found at the base of the nanopipette, which was about 85 µm away from the orifice. The optical microscope image and I-t curve are shown in Fig.4A and Fig.4B. This control experiment revealed the important role of 3.5 nm GNP seeds in the gold deposition at the nanopipette apex. In another control experiment, 20 nm GNP (0.81 nM) was added in the bath solution. The optical microscope image of the gold coated nanopipette is shown in Fig.4C. The current change had the same trend with that using 3.5 nm GNP seeds (Fig.4D). However, it took more than 4 times longer time to deposit gold and fill the barrel at the tip. The length of the
Effects of gold deposition conditions
We investigated various growth parameters affecting the gold deposition at the nanopipette tip. Because the gold deposition was the result of the reduction of AuCl4– ion, we firstly investigated the effect of HAuCl4 concentration on the growth length of gold. While the concentration of NH2OH∙HCl remained at 25 mM, the concentration ratios of NH2OH∙HCl to HAuCl4 were 2:1, 1:1 and 1:2, respectively. After gold deposition at zero bias, the corresponding gold growth lengths were measured to be about 4, 6 and 8 μm by the optical microscopy (Fig.3A‒Fig.3C). Therefore, as expected, the length of grown gold inside the nanopore increases with the increase of chloroauric acid concentration. We then compared the gold deposition with and without
Fig.3 (A‒C) Concentration ratios between NH2OH∙HCl and HAuCl4 are 2:1 (A), 1:1 (B) and 1:2 (C); Optical microscope images of gold nanoelectrode with (D) and without (E) GNPs in the bath solution at zero bias; (F) The nanopipette was immersed in GNPs solution for a few minute with an open circuit
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
Fig.4
Optical microscope image (A) and corresponding i-t curves (B) of gold deposition without GNP seeds in bath solution; Optical microscope image (C) and i-t curve (D) of gold deposition with 20 nm GNPs (0.81 nM) in the bath solution
filled gold was much shorter than that using 3.5 nm GNP seeds. The GNPs with a diameter of 3.5 nm were more beneficial for the formation of gold layer. This was because the presence of big GNPs was often induced further nucleation rather than growth. Therefore, the small GNP seeds were more suitable to realize the particle enlargement method. It should be noted that the circuit must be closed to allow the flow of electrochemical current although the applied bias is zero. To illustrate that the formation of gold at the tip required the application of an electric field, the nanopipette was immersed into GNPs solution under the broken circuit. After a few minutes, the gold blocked the nanopore incompletely and the length of the deposited gold near the tip was only about 2 μm, as shown in Fig.3F. To understand the effect of the applied bias, we also applied +50 and –50 mV biases during gold deposition while keeping other conditions the same. When a +50 mV bias was applied, the ionic current became positive at the beginning (Fig.5A). In the bath solution, GNP seeds with negative charge were attracted into the barrel of nanopipette by the electrostatic force. However, in the barrel, the AuCl4– was driven away from the tip due to the positive bias. The gold deposition at the nanopore was thus limited by the reduced local concentration of AuCl4– near the tip. With the deposition of gold at the inner surface of nanopipette tip, the positive ionic current gradually decreased until became zero. The optical microscope image of the deposited gold at the tip is shown in Fig.5B. The length of deposited gold at the tip was shorter than that at zero bias (Fig.1C). In contrast, at –50 mV, a negative ionic current appeared at the beginning (Fig.5C). The magnitude of the maximum
negative current was normally bigger than that at zero bias. The negative current gradually rose and became positive, reflecting the reduced size of nanopore due to gold deposition and the adsorption of NH3+OH ions at the deposited gold surface. The positive current never went to zero, suggesting that the amount of deposition gold was low and could not fully block the nanopore. In the bath solution, the GNP seeds were repelled away from the orifice of the nanopipette tip. In the barrel, the AuCl4– ions were driven to the tip but the NH3+OH ions were driven away from the tip under the electrostatic force. Therefore, only a small portion of NH3+OH ions successfully met and reacted with AuCl4– ions at the surface of the GNP seeds near the orifice. Most NH3+OH ions were driven away from the tip, creating a depletion zone for NH3+OH ions. Indeed, a void with several-micron long appeared at the tip (Fig.5D). As shown in Fig.5, the gold deposition is highly sensitive to the application of small biases. Overall, these control experiments suggested that the best condition for the growth of gold was at zero bias in the presence of small size GNP seeds in the bath solution and enough AuCl4– ions in the barrel. 3.4
Resistive-pulse measurement of GNP translocation by the gold coated nanopore
To evaluate the performance of gold coated nanopore, the ionic current changes of the nanopore induced by individual GNP translocation events at 800 mV voltage was measured. The negatively charged GNPs with diameter of 20 nm were used in the experiments.
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
Fig.5
(A, B) i-t curve during gold deposition (A) and the corresponding optical microscope images of the deposited gold at the nanopipette tip (B) at +50 mV; (C, D) i-t curve (C) and the optical image (D) at –50 mV
Before adding GNPs in the PBS bath solution (10 mM), the i-t trace is featureless (Fig.6A). After adding GNPs (0.51 nM) in the bath solution, downward ionic current spikes appeared
Fig.6
in the i-t traces (Fig.6B). These current spikes are the transient blocking current induced by the translocation of individual GNPs through the nanopore. Based on the shape of spikes,
i-t curves of nanopore before (A) and after (B) adding 20 nm GNPs at 800 mV voltage; (C) Two zoom-in current spikes; (D) Scatter plot of the current blockade (∆i/i0) versus dwell time (Δt) of GNP translocation signals. The histograms of ∆i/i0 and Δt are shown at the top and right sides, respectively. The solid lines in the histograms are Gaussian fits. The measurements were performed in 10 mM PBS bath solution (N = 57). The scheme shows the movement of the GNPs in the nanopipette under the applied potential. (F) The SEM image of a nanopipette with about 45 nm pore diameter. i-t curves of bare nanopore before (G) and after (H) adding 20 nm GNPs in the bath solution at +800 mV voltage. The solutions in the bath and inside the nanopipette are both 10 mM PBS
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
these measurements results supported that the gold coated nanopipette was used as a resistive-pulse sensor to record the change of current when a single GNP translocated into the nanopore under the influence of electric fields. Figure 6C shows two zoom-in current spikes in Fig.6B. The definition of current spike height (Δi) and dwell time (Δt) is illustrated. Figure 6D displays a scatter plot of Δi/i0 versus Δt of 57 current spikes. I0 is the baseline current, which is typically around 10 pA. The corresponding Δi/i0 and Δt histograms are shown at the top and right sides, respectively. From the Gaussian fits, the mean value of Δt was (0.2 ± 0.3) s and the average Δi/i0 was 1.8 ± 0.8. The scheme shows the movement of the GNPs in the nanopipette under the applied potential (Fig.6E). To fabricate the nanopipette with a smaller nanopore diameter, the quartz capillary tube was pulled with parameters 2. The diameter of bare nanopipette was about 45 nm, as shown in the SEM image in Fig.6F. This size was comparable to the pore size of gold coated nanopipette. Before adding GNPs in the 10 mM PBS bath solution, the i-t trace is featureless (Fig.6G). After adding 0.51 nM GNPs in the bath solution, small current spikes were observed at 800 mV as shown in Fig.6H. The current spikes indicated the GNPs translocation events. Most spikes were downward. However, there were also a few upward spikes. This phenomenon was often observed at low salt concentration. The mean dwell time Δt was (65 ± 12) ms and the corresponding mean normalized spike height Δi/i0 was 0.025 ± 0.004 for the current spikes. Both values were significantly smaller than that of the gold coated nanopipette. Therefore, the gold surface of gold coated nanopipette slowed down the translocation movement of GNPs and increased the induced current changes, both contributing to the greatly improved signal-to-noise ratio (SNR) of the current spike signals as shown in Fig.6B. 3.5
Gold coated nanopipette as a wireless bipolar nanoelectrode
Although unwired, the floated gold layer at the inner surface of the nanopipette can be used as a wireless bipolar nanoelectrode in electrolyte for electrochemical measurements[31]. As shown in Fig.7A, the barrel of nanopipette was filled with 3 M KCl solution containing 5 mM K3Fe(CN)6. The nanopipette was immersed in the bath solution, 0.1 M KCl solution with 2 mM FcMeOH. The Fc in the bath solution was oxidized at a high positive potential. The electrons acquired from the Fc oxidation then transported to the other side of the floating gold inside the barrel, and were consumed for the reduction of Fe(CN)63–. The red curve in Fig.7B is the bipolar CV curve of a wireless gold coated nanopipette. The E1/2 of the CV was about –0.2 V versus Ag/AgCl electrode. For comparison, the bipolar CV measurements in the same solutions were performed by using a
gold nanoelectrode prepared by electrochemical etching of a gold wire[33]. The diagram of the experimental setup is shown in Fig.7C. One GNE was placed in a 3 M KCl solution containing 5 mM ferricyanide, and the other GNE was placed in 0.1 M KCl solution with 2 mM FcMeOH. The two GNEs were electrically connected to serve as a bipolar electrode. The E1/2 values of the both CVs were at the similar potential, suggesting that the gold coated nanopipette could be used as bipolar nanoelectrode. The slope of the red CV near –0.2 V was smaller than that of black CV, reflecting the reduced electron transfer rate at the electrochemically deposited gold surface. For comparison, the standard CV of FcMeOH (blue CV) (2 mM FcMeOH in 0.1 M KCl) was measured by using a wired gold nanoelectrode as the working electrode. The E1/2 was at +0.15 V versus Ag/AgCl electrode. Compared with the standard CV, there was about 350 mV shift in the E1/2 value for the CVs from both bipolar electrodes. These results suggested that the gold coated nanopipette could be used as a wireless bipolar nanoelectrode for catalytic studies or biosensing applications. 3.6
SERS measurement of chemically modified gold ring nanoelectrode
We further tested the capability of the integrated gold nanoring electrode of the nanopipette as a SERS substrate. SERS can provide molecular fingerprints, which is important to recognize molecules without ambiguity. The SERS spectra of 4-ATP have been extensively studied[34]. We therefore used 4-ATP as model molecule here. The 4-ATP molecules were self-assembled on the gold ring nanoelectrode. The thiol group of 4-ATP was bound with gold through Au–S covalent bond and the amine group of 4-ATP orientated upward. Figure 8A shows the I-V curves of gold coated nanopipette before (curve a) and after (curve b) 4-ATP modification. After modification with 4-ATP, the ionic current obviously reduced. In addition, the I-V curve was more symmetric and the rectification ratio[35] (|I(–0.4 V)/I(+0.4 V)|) was reduced from 3.34 to 1.43. These changes indicated that the 4-ATP had been successfully modified on the gold surface. The immobilized 4-ATP molecules suppressed the ionic current and reduced the negative surface charge of nanopipette inner surface. In the next step, the nanopipette tip was functionalized with 50 nm AgNPs. The AgNPs were immobilized at the 4-ATP modified gold surface mainly through the electrostatic attractive forces. The optical fields were greatly enhanced in the Au-4-ATP-AgNPs nanogap because of the formation of ‘hotspots’, which significantly boosted the Raman signals. A typical SERS spectrum is shown in Fig.8B, with spectra region between 900 and 1700 cm–1. The two fingerprint peaks of 4-ATP can be identified in the spectrum. The peak at 1083 cm–1 is assigned to the stretching mode of C–S bond coupled with benzene ring; and the peak at 1580 cm–1 is assigned to the
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
Fig.7
(A) Schematic diagram of bipolar electrochemistry experiment using gold coated nanopipette; (B) Bipolar voltammetric response of gold coated nanopipette in 2 mM FcMeOH and 0.1 M KCl. The red CV is the bipolar electrode filled with 3 M KCl and 5 mM K3Fe(CN)6. The black CV curve is the normalized response by using etched gold nanoelectrodes at the same solutions. The blue CV curve is the standard CV of FcMeOH using a wired gold nanoelectrode; (C) Diagram of the control experimental setup Δ
Fig.8
(A) I-V curves before (a) and after (b) surface modification of 4-ATP in 10 mM PBS solution (pH 7.69); (B) SERS spectrum of Au-4-ATP-Ag NPs
stretching mode of Cring–Cring bond. In addition, three pronounced peaks appear between 1053 and 1580 cm–1. These peaks are likely from 4,4′-dimercaptoazobenzene (DMAB) because of the metal NP induced catalytic reaction of 4-ATP in the plasmonic junctions[36]. The peak at 1142 cm–1 is assigned to the bending of C–H bond; peaks at 1392 cm–1 and 1433 cm–1 are assigned to the stretching of N=N. The broadening of peak at 1580 cm–1 is also due to the conversion from 4-ATP to DMAB. The lower intensity of the 1083 cm‒1 peak than the DMAB related peaks suggests that the conversion from 4-ATP to DMAB is very effective in the plasmonic junctions formed by the adsorbed Ag NPs at the apex of the gold coated nanopipette. These results suggested that the gold coated nanopipette could be used as an effective SERS substrate.
gold surface, we expected that the further chemical modification of the gold surface could effectively control the translocation events of single entities. Further, the fabricated wireless nanoelectrode could also be used as the bipolar electrode for electrochemical measurement and as a substrate for SERS. The metalized nanopipette will enable multifunctional single-entity detection by both nanoscale electrochemical measurement and SERS during the translocation events at the nanopipette tip.
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Cite this article as: Chinese J. Anal. Chem., 2019, 47(8): e19104–e19112
RESEARCH PAPER
Facile Fabrication of Gold Functionalized Nanopipette for Nanoscale Electrochemistry and Surface Enhanced Raman Spectroscopy LI Hong-Na, YANG Dan, LIU Ao-Xue, LIU Guo-Hui, SHAN Yu-Ping, YANG Guo-Cheng*, HE Jin* School of Chemistry and Life Science, Advanced Institute of Materials Science, Changchun University of Technology, Changchun 130012, China
Abstract:
Nanopipette has emerged as a versatile nanosensor. It is desirable to expand the sensing capabilities of nanopipette by
integrating multiple sensing methods into one nanopipette. In this work, we presented a seeded growth approach to facilely, cheaply and quickly deposit a gold layer with controllable length at the inner surface of the nanopipette tip. The deposited gold formed a wireless ring-shape gold nanoelectrode at the nanopipette tip and enabled multimode single entity detection. With the presence of gold at the nanopipette apex, the nanopore based resistive-pulse sensing capability was obviously improved. The integrated ring-shape gold nanoelectrode could be used as a wireless bipolar electrode for electrochemical measurement. We further demonstrated that the gold coated nanopipette could be used as a substrate for surface enhanced Raman spectroscopy. Key Words:
Nanopipette; electrochemical method; Bipolar nanoelectrode; Raman spectroscopy
1 Introduction Nanopore technology provides a promising method to monitor the dynamics of single entity in real-time in ionic solutions. As a well-established single-entity nanosensor, it has been extensively used in applications such as DNA sequencing[1,2], single protein analysis[3,4] and single nanoparticle detection[5,6]. The basic nanopore sensing method is based on the resistive-pulse sensing[7]. A constant voltage is applied across the nanopore to drive the charged molecule or nanoparticle to move through a nanopore with a comparable size. The single-entity translocation event causes a transient ionic current change in the current-time trace. It is critical to fabricate size-controllable single nanopore for the analysis and detection of a ranges of nanoscale objects. Nanopipette is a special type of nanopore with a conical shape. Different from other solid-state nanopores, the fabrication of quartz/glass nanopipette with variable pore sizes is cheap, simple and reproducible. Nanopipettes have been developed
as effective chemical and biological sensors and exhibit a sensitivity at single-molecule level for DNA and protein studies[8,9]. Uniquely, the nanopipette can also be conveniently used for single living cell analysis[10,11]. It is of great interest to use nanopipette as a functional biosensor. However, it is still challenging for detection of analyte in a complicated environment, such as cytoplasm and extracellular fluid. To improve its sensitivity and selectivity, previous studies modified the nanopipette inner surface with carbon[12‒14], metal[15] or recognition molecules[16,17], while keeping the nanopore open. One approach is to fabricate metalized nanopipettes to integrate different sensing modalities, which can enhance both the sensitivity and selectivity of the nanopipette sensor. Various methods have been developed to fabricate nanoelectrodes, such as electron beam evaporation[18] to fabricate metallic nanoelectrode, chemical vapor deposition (CVD)[19] to fabricate carbon nanoelectrode, and laser pulling method[20] to fabricate a platinum or gold nanoelectrode. The conductive nanotip has
________________________ Received 18 March 2019; accepted 7 May 2019 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 21673023, 21773017). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61177-1
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
been investigated and reported by several groups [21,22]. For example, the work of nanoelectrode fabrication reported by Hao et al[23] suggested a new electrochemically plating approach by using liquid gallium/indium alloy to deposit gold on the nanopore of nanopipette. However, the fabrication process needs focused ion beam (FIB) milling method to expose the fresh metal nanoelectrode after gold deposition, which is time consuming and the yield is low. Xu et al[24] used a facile one-step photochemical approach to deposit an ultrathin gold film on the inner surface of the nanopipette tip. However, the deposition process is still very slow. In this work, we developed a gold nanoparticle (GNP) seed based electrochemical deposition method to deposit gold on the inner surface of the nanopipette tip. The gold deposition speed is fast, and the deposition process is self-terminated in a few minutes. After thermal annealing, ring-shape gold nanoelectrode and gold coated nanopore are formed at the nanopipette tip. By using GNPs as the model analyte, the gold coated nanopore shows improved signal-to-noise ratio (SNR) for the nanopore based resistive-pulse sensing method. In addition, the presence of gold at the nanopipette tip introduces new functions. The gold nanoelectrode is suitable for nanoscale electrochemical measurements and surface enhanced Raman spectroscopy (SERS). Besides, the ringshape gold at the nanopipette tip can be used as a bipolar nanoelectrode and as an effective substrate for SERS.
2
Experimental
2.1
Chemicals
Potassium (KCl) and sodium chloride (NaCl) were purchased from Beijing Chemical Works. Sulfuric acid (H2SO4), hydrogen peroxide (H2O2), sodium borohydride (NaBH4), and trisodium citrate (Na3C6H5O7∙2H2O) were purchased from Sinopharm Group. Gold chloride (HAuCl4∙xH2O) was purchased from Macklin. Hydroxylamine hydrochloride (NH2OH∙HCl) was purchased from Xilong Chemical Co., Ltd. Potassium ferricyanide (K3Fe(CN)6), ferrocenemethanol (FcMeOH), 4-aminothiophenol (4-ATP), monopotassium phosphate (KH2PO4), disodium phosphate (Na2HPO4) and sodium hypochlorite were purchased from Aladdin. The solutions were prepared using deionized (DI) water (18.2 MΩ cm at 25 ºC) from a water purification system (Millipore S.A.S). All reagents and materials were of at least analytical grade. The 50 nm silver nanoparticles (AgNPs) were prepared by reduction of silver nitrate with trisodium citrate using a well-established method[25]. 2.2
Fabrication of nanopipette
The
quartz
capillary
tubes
with
filament
(FG-G
QT100-50-7.5, Sutter Instrument) were used to fabricate the gold nanoelectrode. The pipettes were first cleaned by Piranha (the volume ratio of H2SO4 to H2O2 is 3:1, caution: Piranha solutions are highly corrosive and need to be handled with extreme caution.) for 30 min, then repeatedly rinsed with DI water and dried in an oven at 100 °C for overnight. The quartz nanopipettes with nanopore diameter of about 100 nm were fabricated from these cleaned capillary tubes by using a laser pipette puller (P-2000, Sutter Instrument) with following parameters 1: HEAT = 650, FIL = 4, VEL = 60, DEL = 145, PUL = 100. The quartz capillary tube is pulled with the following parameters 2: HEAT = 700, FIL = 3, VEL = 40, DEL = 175, PUL = 190 to fabricate the nanopore with about 45 nm diameter. 2.3
Measurements
Cyclic voltammogram (CV) and current-time (i-t) traces during gold deposition were acquired with a CHI852c electrochemical workstation. The Ag/AgCl wire electrode was used as the quasi-reference electrode in all electrochemical experiments. Platinum wire was used as the counter electrode for CV measurements. A scan rate of 50 mV s‒1 was used for all CV curves. The current-voltage (i-V) curves were measured by using a low noise source measure meter (2636B Keithley). For the resistive-pulse measurement of nanopipette, the i-t traces for nanoparticle translocation experiments were measured by using Synthesized Function Generatou (Model DS345) to prove voltage and the corresponding data were recorded by an Axon digidata 1550B. Two Ag/AgCl wire electrodes were used as the electrodes. The electrode in the PBS (10 mM, pH 7.4) bath solution was always grounded and the Vpore was applied at the Ag/AgCl electrode inside the nanopipette barrel. The traces were recorded at a 15 kHz sampling rate with a 30 Hz low-pass filter. All the measurements were performed at room temperature (25 °C). The scanning electron microscopy (SEM) images of nanopipettes were obtained by a field-emission SEM instrument (JOEL7610) and the samples were coated with a thin Pt layer before imaging. The optical microscope images were acquired by a Nikon Ti-U inverted microscope with a 40× objective lens. 2.4
Preparation of GNP
GNP seeds with diameter of about 3.5 nm were synthesized by using a strong reducing reagent (NaBH4) according to the literature method[26]. To synthesize 20-nm size GNPs for the control experiment, the AuCl4– ion was reduced by trisodium citrate[27]. The concentrations of 3.5-nm GNPs and 20-nm GNPs in the original solutions were 51 and 0.81 nM, respectively[28].
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
2.5
SERS measurement
The gold coated nanopipette was immersed in 4-ATP ethanol solution (50 mM) for 12 h and then rinsed copiously by DI water. In the next step, the 4-ATP modified gold coated nanopipette was immersed in 50-nm AgNP solution (0.225 nM) for 4 h. The Raman spectrum analysis was performed on a Horiba LabRam HR Evolution Raman spectrometer with 532-nm laser line and exposure time of 10 s.
3 Results and discussion 3.1
Fabrication of gold coated nanopipette
NaBH4 as a strong reductive reagent is widely used for the reduction of AuCl4– ion. However, this reaction occurs at a violent reaction rate and the generation of hydrogen gas can cause many defective cavities in the deposited gold [29,30]. To avoid this problem, we instead use a mild reductive agent NH2OH∙HCl to deposit gold on the tip of nanopipette. Wang et al[31] reported a gold deposition method based on a mild reducing reagent (NH2OH∙HCl). However, this gold deposition process is extremely slow, which will last three days. Different from previous methods, we added small GNPs with diameter about 3.5 nm in the DI water as the seeds. As we discussed later, the GNP seeds in the bath solution greatly speeded up the gold deposition process at the nanopipette tip. The setup is shown in Fig.1A. The nanopipette barrel was filled with a mixture solution of NH2OH∙HCl and HAuCl4 at concentration ratio of 1:2. The concentration of NH2OH∙HCl was 25 mM. The bath solution contained 51 nM 3.5 nm GNP seeds. We maintained a zero bias between two Ag/AgCl quasi reference electrodes and monitored the current change during gold deposition. The chemical reaction during gold deposition
process can be expressed as: 6NH2OH + 2HAuCl4 = 2Au (s) + 3N2 (g) + 8HCl + 6H2O This reaction mainly occurs at the nanopipette orifice, where the bath solution and the barrel solution are mixed. Figure 1B shows a typical i-t trace during gold deposition. Based on the current change, the gold deposition is divided into three stages. In the first stage, a negative current is quickly established right after the nanopipette tip is immersed in the bath solution to close the circuit. The negative ionic current is generated because of the concentration gradient and the negative surface charge of nanopipette, which driving the AuCl4– ions inside the nanopipette barrel to move out of the nanopipette apex. The gold deposition process, starting from the nanopipette apex, is accelerated by the GNP seeds, which serving as the nucleation centers[32]. In stage II, the negative current gradually rises, becomes positive and reaches a positive maximum. This is attributed to the gradual adsorption of cations, such as NH3+OH, to the negatively charged inner wall surface. The surface charge becomes positive and the positive surface potential drives a positive ionic current. The negatively charged GNP seeds in the bath solution can also be brought into the barrel under a positive potential. At stage III, the positive current falls continuously until reaches zero. The decrease of ionic current is induced by the shrinkage of pore size due to gold deposition. At zero current, the barrel at the nanopipette apex is fully clogged and the gold deposition is terminated automatically. The whole deposition progress generally only lasts a few minutes. The gold deposition at the tip can be clearly revealed in the optical microscope image, as shown in Fig.1C. The length of the deposited gold is about 7–8 μm. After electrochemical deposition, the nanopipette was cleaned and annealed in air at 300 °C for 4 h in a muffle furnace to improve the quality of deposited gold and the adhesion between the gold and the nanopipette inner surface.
Fig.1 (A) Experimental setup for gold deposition on inner surface of nanopipette tip. The bath solution is DI water with GNP seeds (51 nM); (B) Electrochemical i-t trace during the gold deposition at nanopipette tip. (C) Optical microscope image of the deposited gold at the tip of nanopipette
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
Fig.2
SEM images of nanopipette before (A) and (B) after gold deposition and annealing. (C) I-V curves of bare nanopipette (a), before (b) and after (c) annealing at 300 °C for 4 h. I-V curves are measured in PBS solution (10 mM)
To optimize and understand the self-terminated seed assisted gold deposition mechanism, different growth conditions were optimized. The gold deposition at different conditions is discussed later. Especially, the gold deposition is very sensitive to the external bias, which regulates the movement of ions through the nanopore and controls the reaction rate. 3.2
Characterization of bare nanopipette and gold coated nanopipette
SEM images of bare nanopipette and gold coated nanopipette are shown in Fig.2A and Fig.2B, respectively. Before gold deposition, the diameter of the bare nanopipette was about 100 nm. After annealing the deposited gold, the pore diameter of gold coated nanopipette was reduced to about 40 nm. The typical I-V curves measured in PBS solution (10 mM) are shown in Fig.2C. After gold deposition, the current was almost zero in the I-V curve (green curve), suggesting the pore was almost fully blocked. However, after annealing, the current through the nanopipette was partially recovered (red curve), which indicated the formation of a ring shape gold nanoelectrode at the nanopipette tip. These measurement results suggested that the seed growthbased electrochemical deposition approach was successful and a ring shape gold nanoelectrode was formed after annealing. 3.3
3.5 nm GNP seeds in the bath solution while fixing the concentration ratio of NH2OH∙HCl to HAuCl4 of nanopipette barrel solution at 1:2. The optical microscope images of gold coated nanopipette with and without GNP seeds in the bath solution are shown in Figs. 3D and 3E respectively. With the 3.5 nm GNP seeds in the bath solution, the gold deposition began from the orifice of the nanopipette and extends a length of 7–8 μm inside the barrel (Fig.3D). The GNP seeds were accumulated towards the nanopipette tip and worked as nucleation centers to trigger gold deposition right from the orifice, where the bath and barrel solutions mixed. In contrast, without GNP seeds in the bath solution, no significant gold deposition was observed at the orifice of the nanopipette. The length of gold layer was only about 1 μm at the tip (Fig.3E). Most of gold aggregates in 5-µm size were only found at the base of the nanopipette, which was about 85 µm away from the orifice. The optical microscope image and I-t curve are shown in Fig.4A and Fig.4B. This control experiment revealed the important role of 3.5 nm GNP seeds in the gold deposition at the nanopipette apex. In another control experiment, 20 nm GNP (0.81 nM) was added in the bath solution. The optical microscope image of the gold coated nanopipette is shown in Fig.4C. The current change had the same trend with that using 3.5 nm GNP seeds (Fig.4D). However, it took more than 4 times longer time to deposit gold and fill the barrel at the tip. The length of the
Effects of gold deposition conditions
We investigated various growth parameters affecting the gold deposition at the nanopipette tip. Because the gold deposition was the result of the reduction of AuCl4– ion, we firstly investigated the effect of HAuCl4 concentration on the growth length of gold. While the concentration of NH2OH∙HCl remained at 25 mM, the concentration ratios of NH2OH∙HCl to HAuCl4 were 2:1, 1:1 and 1:2, respectively. After gold deposition at zero bias, the corresponding gold growth lengths were measured to be about 4, 6 and 8 μm by the optical microscopy (Fig.3A‒Fig.3C). Therefore, as expected, the length of grown gold inside the nanopore increases with the increase of chloroauric acid concentration. We then compared the gold deposition with and without
Fig.3 (A‒C) Concentration ratios between NH2OH∙HCl and HAuCl4 are 2:1 (A), 1:1 (B) and 1:2 (C); Optical microscope images of gold nanoelectrode with (D) and without (E) GNPs in the bath solution at zero bias; (F) The nanopipette was immersed in GNPs solution for a few minute with an open circuit
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
Fig.4
Optical microscope image (A) and corresponding i-t curves (B) of gold deposition without GNP seeds in bath solution; Optical microscope image (C) and i-t curve (D) of gold deposition with 20 nm GNPs (0.81 nM) in the bath solution
filled gold was much shorter than that using 3.5 nm GNP seeds. The GNPs with a diameter of 3.5 nm were more beneficial for the formation of gold layer. This was because the presence of big GNPs was often induced further nucleation rather than growth. Therefore, the small GNP seeds were more suitable to realize the particle enlargement method. It should be noted that the circuit must be closed to allow the flow of electrochemical current although the applied bias is zero. To illustrate that the formation of gold at the tip required the application of an electric field, the nanopipette was immersed into GNPs solution under the broken circuit. After a few minutes, the gold blocked the nanopore incompletely and the length of the deposited gold near the tip was only about 2 μm, as shown in Fig.3F. To understand the effect of the applied bias, we also applied +50 and –50 mV biases during gold deposition while keeping other conditions the same. When a +50 mV bias was applied, the ionic current became positive at the beginning (Fig.5A). In the bath solution, GNP seeds with negative charge were attracted into the barrel of nanopipette by the electrostatic force. However, in the barrel, the AuCl4– was driven away from the tip due to the positive bias. The gold deposition at the nanopore was thus limited by the reduced local concentration of AuCl4– near the tip. With the deposition of gold at the inner surface of nanopipette tip, the positive ionic current gradually decreased until became zero. The optical microscope image of the deposited gold at the tip is shown in Fig.5B. The length of deposited gold at the tip was shorter than that at zero bias (Fig.1C). In contrast, at –50 mV, a negative ionic current appeared at the beginning (Fig.5C). The magnitude of the maximum
negative current was normally bigger than that at zero bias. The negative current gradually rose and became positive, reflecting the reduced size of nanopore due to gold deposition and the adsorption of NH3+OH ions at the deposited gold surface. The positive current never went to zero, suggesting that the amount of deposition gold was low and could not fully block the nanopore. In the bath solution, the GNP seeds were repelled away from the orifice of the nanopipette tip. In the barrel, the AuCl4– ions were driven to the tip but the NH3+OH ions were driven away from the tip under the electrostatic force. Therefore, only a small portion of NH3+OH ions successfully met and reacted with AuCl4– ions at the surface of the GNP seeds near the orifice. Most NH3+OH ions were driven away from the tip, creating a depletion zone for NH3+OH ions. Indeed, a void with several-micron long appeared at the tip (Fig.5D). As shown in Fig.5, the gold deposition is highly sensitive to the application of small biases. Overall, these control experiments suggested that the best condition for the growth of gold was at zero bias in the presence of small size GNP seeds in the bath solution and enough AuCl4– ions in the barrel. 3.4
Resistive-pulse measurement of GNP translocation by the gold coated nanopore
To evaluate the performance of gold coated nanopore, the ionic current changes of the nanopore induced by individual GNP translocation events at 800 mV voltage was measured. The negatively charged GNPs with diameter of 20 nm were used in the experiments.
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
Fig.5
(A, B) i-t curve during gold deposition (A) and the corresponding optical microscope images of the deposited gold at the nanopipette tip (B) at +50 mV; (C, D) i-t curve (C) and the optical image (D) at –50 mV
Before adding GNPs in the PBS bath solution (10 mM), the i-t trace is featureless (Fig.6A). After adding GNPs (0.51 nM) in the bath solution, downward ionic current spikes appeared
Fig.6
in the i-t traces (Fig.6B). These current spikes are the transient blocking current induced by the translocation of individual GNPs through the nanopore. Based on the shape of spikes,
i-t curves of nanopore before (A) and after (B) adding 20 nm GNPs at 800 mV voltage; (C) Two zoom-in current spikes; (D) Scatter plot of the current blockade (∆i/i0) versus dwell time (Δt) of GNP translocation signals. The histograms of ∆i/i0 and Δt are shown at the top and right sides, respectively. The solid lines in the histograms are Gaussian fits. The measurements were performed in 10 mM PBS bath solution (N = 57). The scheme shows the movement of the GNPs in the nanopipette under the applied potential. (F) The SEM image of a nanopipette with about 45 nm pore diameter. i-t curves of bare nanopore before (G) and after (H) adding 20 nm GNPs in the bath solution at +800 mV voltage. The solutions in the bath and inside the nanopipette are both 10 mM PBS
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
these measurements results supported that the gold coated nanopipette was used as a resistive-pulse sensor to record the change of current when a single GNP translocated into the nanopore under the influence of electric fields. Figure 6C shows two zoom-in current spikes in Fig.6B. The definition of current spike height (Δi) and dwell time (Δt) is illustrated. Figure 6D displays a scatter plot of Δi/i0 versus Δt of 57 current spikes. I0 is the baseline current, which is typically around 10 pA. The corresponding Δi/i0 and Δt histograms are shown at the top and right sides, respectively. From the Gaussian fits, the mean value of Δt was (0.2 ± 0.3) s and the average Δi/i0 was 1.8 ± 0.8. The scheme shows the movement of the GNPs in the nanopipette under the applied potential (Fig.6E). To fabricate the nanopipette with a smaller nanopore diameter, the quartz capillary tube was pulled with parameters 2. The diameter of bare nanopipette was about 45 nm, as shown in the SEM image in Fig.6F. This size was comparable to the pore size of gold coated nanopipette. Before adding GNPs in the 10 mM PBS bath solution, the i-t trace is featureless (Fig.6G). After adding 0.51 nM GNPs in the bath solution, small current spikes were observed at 800 mV as shown in Fig.6H. The current spikes indicated the GNPs translocation events. Most spikes were downward. However, there were also a few upward spikes. This phenomenon was often observed at low salt concentration. The mean dwell time Δt was (65 ± 12) ms and the corresponding mean normalized spike height Δi/i0 was 0.025 ± 0.004 for the current spikes. Both values were significantly smaller than that of the gold coated nanopipette. Therefore, the gold surface of gold coated nanopipette slowed down the translocation movement of GNPs and increased the induced current changes, both contributing to the greatly improved signal-to-noise ratio (SNR) of the current spike signals as shown in Fig.6B. 3.5
Gold coated nanopipette as a wireless bipolar nanoelectrode
Although unwired, the floated gold layer at the inner surface of the nanopipette can be used as a wireless bipolar nanoelectrode in electrolyte for electrochemical measurements[31]. As shown in Fig.7A, the barrel of nanopipette was filled with 3 M KCl solution containing 5 mM K3Fe(CN)6. The nanopipette was immersed in the bath solution, 0.1 M KCl solution with 2 mM FcMeOH. The Fc in the bath solution was oxidized at a high positive potential. The electrons acquired from the Fc oxidation then transported to the other side of the floating gold inside the barrel, and were consumed for the reduction of Fe(CN)63–. The red curve in Fig.7B is the bipolar CV curve of a wireless gold coated nanopipette. The E1/2 of the CV was about –0.2 V versus Ag/AgCl electrode. For comparison, the bipolar CV measurements in the same solutions were performed by using a
gold nanoelectrode prepared by electrochemical etching of a gold wire[33]. The diagram of the experimental setup is shown in Fig.7C. One GNE was placed in a 3 M KCl solution containing 5 mM ferricyanide, and the other GNE was placed in 0.1 M KCl solution with 2 mM FcMeOH. The two GNEs were electrically connected to serve as a bipolar electrode. The E1/2 values of the both CVs were at the similar potential, suggesting that the gold coated nanopipette could be used as bipolar nanoelectrode. The slope of the red CV near –0.2 V was smaller than that of black CV, reflecting the reduced electron transfer rate at the electrochemically deposited gold surface. For comparison, the standard CV of FcMeOH (blue CV) (2 mM FcMeOH in 0.1 M KCl) was measured by using a wired gold nanoelectrode as the working electrode. The E1/2 was at +0.15 V versus Ag/AgCl electrode. Compared with the standard CV, there was about 350 mV shift in the E1/2 value for the CVs from both bipolar electrodes. These results suggested that the gold coated nanopipette could be used as a wireless bipolar nanoelectrode for catalytic studies or biosensing applications. 3.6
SERS measurement of chemically modified gold ring nanoelectrode
We further tested the capability of the integrated gold nanoring electrode of the nanopipette as a SERS substrate. SERS can provide molecular fingerprints, which is important to recognize molecules without ambiguity. The SERS spectra of 4-ATP have been extensively studied[34]. We therefore used 4-ATP as model molecule here. The 4-ATP molecules were self-assembled on the gold ring nanoelectrode. The thiol group of 4-ATP was bound with gold through Au–S covalent bond and the amine group of 4-ATP orientated upward. Figure 8A shows the I-V curves of gold coated nanopipette before (curve a) and after (curve b) 4-ATP modification. After modification with 4-ATP, the ionic current obviously reduced. In addition, the I-V curve was more symmetric and the rectification ratio[35] (|I(–0.4 V)/I(+0.4 V)|) was reduced from 3.34 to 1.43. These changes indicated that the 4-ATP had been successfully modified on the gold surface. The immobilized 4-ATP molecules suppressed the ionic current and reduced the negative surface charge of nanopipette inner surface. In the next step, the nanopipette tip was functionalized with 50 nm AgNPs. The AgNPs were immobilized at the 4-ATP modified gold surface mainly through the electrostatic attractive forces. The optical fields were greatly enhanced in the Au-4-ATP-AgNPs nanogap because of the formation of ‘hotspots’, which significantly boosted the Raman signals. A typical SERS spectrum is shown in Fig.8B, with spectra region between 900 and 1700 cm–1. The two fingerprint peaks of 4-ATP can be identified in the spectrum. The peak at 1083 cm–1 is assigned to the stretching mode of C–S bond coupled with benzene ring; and the peak at 1580 cm–1 is assigned to the
LI Hong-Na et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19104–e19112
Fig.7
(A) Schematic diagram of bipolar electrochemistry experiment using gold coated nanopipette; (B) Bipolar voltammetric response of gold coated nanopipette in 2 mM FcMeOH and 0.1 M KCl. The red CV is the bipolar electrode filled with 3 M KCl and 5 mM K3Fe(CN)6. The black CV curve is the normalized response by using etched gold nanoelectrodes at the same solutions. The blue CV curve is the standard CV of FcMeOH using a wired gold nanoelectrode; (C) Diagram of the control experimental setup Δ
Fig.8
(A) I-V curves before (a) and after (b) surface modification of 4-ATP in 10 mM PBS solution (pH 7.69); (B) SERS spectrum of Au-4-ATP-Ag NPs
stretching mode of Cring–Cring bond. In addition, three pronounced peaks appear between 1053 and 1580 cm–1. These peaks are likely from 4,4′-dimercaptoazobenzene (DMAB) because of the metal NP induced catalytic reaction of 4-ATP in the plasmonic junctions[36]. The peak at 1142 cm–1 is assigned to the bending of C–H bond; peaks at 1392 cm–1 and 1433 cm–1 are assigned to the stretching of N=N. The broadening of peak at 1580 cm–1 is also due to the conversion from 4-ATP to DMAB. The lower intensity of the 1083 cm‒1 peak than the DMAB related peaks suggests that the conversion from 4-ATP to DMAB is very effective in the plasmonic junctions formed by the adsorbed Ag NPs at the apex of the gold coated nanopipette. These results suggested that the gold coated nanopipette could be used as an effective SERS substrate.
gold surface, we expected that the further chemical modification of the gold surface could effectively control the translocation events of single entities. Further, the fabricated wireless nanoelectrode could also be used as the bipolar electrode for electrochemical measurement and as a substrate for SERS. The metalized nanopipette will enable multifunctional single-entity detection by both nanoscale electrochemical measurement and SERS during the translocation events at the nanopipette tip.
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