Asymmetric Nanopore Electrode-Based Amplification for Electron

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Asymmetric Nanopore Electrode Based Amplification for Electron Transfer Imaging in Live Cells Yi-Lun Ying, Yong-Xu Hu, Rui Gao, Ru-Jia Yu, Zhen Gu, Luke P. Lee, and Yi-Tao Long J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b12106 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 13, 2018

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Journal of the American Chemical Society

Asymmetric Nanopore Electrode-Based Amplification for Electron Transfer Imaging in Live Cells Yi-Lun Ying1#, Yong-Xu Hu1#, Rui Gao1, Ru-Jia Yu1, Zhen Gu1, Luke P. Lee2, 3, 4, 5, Yi-Tao Long1* 1 Key Laboratory for Advanced Materials & School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China 2 Biomedical Institute for Global Health Research and Technology, National University of Singapore, 117599, Singapore 3Departments of Bioengineering, Electrical Engineering and Computer Sciences, UC Berkeley, CA 94720, USA 4Berkeley Sensor and Actuator Center, UC Berkeley, CA 94720, USA 5 Biophysics Graduate Program, UC Berkeley, CA 94720, USA KEYWORDS: Nanopore electrode, Glass nanopore, Single cell, Single molecule, NADH

ABSTRACT: Capturing real-time electron transfer, enzyme activity, molecular dynamics, and biochemical messengers in living cells is essential for understanding the signaling pathways and cellular communications. However, there is no generalizable method for characterizing a broad range of redox-active species in a single living cell at the resolution of cellular compartments. Although nanoelectrodes have been applied in the intracellular detection of redox-active species, the fabrication of nanoelectrodes to maximize the signal-to-noise ratio of the probe remains challenging because of the stringent requirements of 3D fabrication. Here, we report an asymmetric nanopore electrode-based amplification mechanism for the real-time monitoring of NADH in a living cell. We used a two-step 3D fabrication process to develop a modified asymmetric nanopore electrode with a diameter down to 90 nm, which allowed for the detection of redox metabolism in living cells. Taking advantage of the asymmetric geometry, the above 90% potential drop at the two terminals of the nanopore electrode converts the faradaic current response into an easily distinguishable bubble-induced transient ionic current pattern. Therefore, the current signal was amplified by at least 3 orders of magnitude, which was dynamically linked to the presence of trace redox-active species. Compared to traditional wire electrodes, this wireless asymmetric nanopore electrode exhibits a high signal-to-noise ratio by increasing the current resolution from nanoamperes to picoamperes. The asymmetric nanopore electrode achieves the highly sensitive and selective probing of NADH concentrations as low as 1 pM. Moreover, it enables the real-time nanopore monitoring of the respiration chain (i.e., NADH) in a living cell and the evaluation of the effects of anticancer drugs in an MCF-7 cell. We believe that this integrated wireless asymmetric nanopore electrode provides promising building blocks for the future imaging of electron transfer dynamics in live cells.

Introduction The real-time monitoring of heterogeneous cellular behavior at the single-cell level is key to understanding the dynamics of metabolism, signaling, ion transport and protein activities in a complex cellular environment.1-2 The electrochemical method is powerful for studying dynamics of redox metabolism in individual cells due to its remarkable advantages of selectivity and sensitivity, its label-free approach, and the direct correlation between the signals and the electroactive substances in cells.3-7 To realize the real-time monitoring of electrochemical redox metabolism in single living cells within the scope of the natural environment, the diameter of the electrode should be less than 100 nm to avoid damaging the function of the single cell. More importantly, a nanoscale electrode allows for high spatial and temporal resolution in single-cell analysis.8-10 The implementation of this approach is largely determined by the electrochemical performance of the nanoelectrode, which endures a complicated, time-consuming and uncontrollable fabrication process. For example, the fabrication of platinum or gold disk nanoelectrodes by laser pulling of glass-sealed metal wires often requires the extremely precise manipulation and

sophisticated polishing of the nanoelectrodes.11-12 This fabrication process may introduce certain defects or surface inhomogeneity on the nanowire of the nanoelectrode, resulting in a poor signal-to-noise ratio with a low current and low temporal resolution.13-14 More importantly, the current method suffers from poor signal responses, which restrict the application of the electrochemical method to certain analytes with high intracellular concentrations (~ 1 µM). Although there have been considerable efforts to develop a variety of nanoelectrodes, there is no general methodology for achieving suitable signal amplification to characterize a broad range of redox active species in live cells. Here, we addressed this need by introducing a new class of asymmetric nanopore electrodes (ANEs), which do not require the sealing of a traditional metal wire inside a nanoelectrode. The wireless ANE is designed to have an Au-coated interior as a sensing interface instead of the traditional electrode (Figure 1a-c). The simulation and characterization of this innovative wireless asymmetric electrode within a nanopore reveal advantages of a simple fabrication technique, easy modification, and controllable dimensions. Owing to the advantages of the

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bipolar interface at both ends of gold layer inside the asym-

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metric nanopore, the redox target can be easily

Figure 1 │ Asymmetric nanopore electrode for monitoring electron transfer dynamics in live cells. (a) Illustration of an asymmetric nanopore electrode (ANE) for probing redox-active species. The applied bias potential (Ebias) drives the potential difference at the two terminals of the Au-coated asymmetric nanopore. At a negative potential, the tip opening (cis) is polarized as a cathode (δ-), while the opposite terminal (trans) acts an anode (δ+). The gradation of the Au layer from red to yellow represents the polarization. Reduction occurs at the cathode, while oxidation occurs at the anode of the ANE. (b) The ANE for single-cell probing. The intracellular redox specie (e.g., NADH) diffuses into the cis tip of the ANE through the tip opening; thus, a pair of redox reactions occur at the cathode (δ-) and anode (δ+). The ANE acquires a faradaic current response at the interface of the Au layer through a distinguishable transient ionic current response pattern with a high current and high temporal resolution. (c) A traditional nanoelectrode (e.g., carbon nanoelectrode) for probing intracellular redox species. The redox reaction occurs at the interface of the solid electrode wire tip, generating a cyclic voltammogram with a low signal-to-noise ratio and poor temporal resolution. (d) The bare nanopore does not produce any transient responses to NADH. (e) The unmodified ANE generates a stable baseline without any signals. (f) The 4-thiol-catechol-modified ANE (CS-ANE) generates an enhanced current signal due to the generation of H2 nanobubbles at the cathodic pole. Continuous current modulations were recorded at -0.7 V. Left: the catechol can be oxidized electrochemically to the corresponding o-benzoquinone at the anodic pole, whereas H2 is generated at the cathodic pole. Right: in the presence of NADH, the o-benzoquinone/catechol conversion is mediated by the redox couple NADH/NAD+, leading to an increased current response with a high amplitude. Ebias is set at – 0.7 V. The electrolyte solution is 10 mM PBS. A pair of Ag/AgCl electrodes is used to apply the bias potential. The electrode close to the tip of the nanopore is defined as the virtual ground. determined via hydrogen-bubble-induced current amplification within a confined nanopore (Figure 1d-f). The generalizable detection mechanism of ANEs provides an ionic current response pattern with a high current and high temporal resolution (Figure 1f), which is important for clearly understanding the signaling pathways and cellular communication in a living cell. Previous works in our group have shown that Ag-coated nanopores could control the diffusion profile of Ag+.15-16 Therefore, the ANEs further achieve the control and amplification of the unmeasurable current response of nicotinamide adenine nucleotide (NADH) into a clearly pulse-like current signal by at least 3 orders of magnitude, allowing for the real-time monitoring of NADH concentrations as low as 1 pM. To the best of our knowledge, this is the first electrochemical report of the real-time monitoring of the respiration chain (i.e., NADH) inside a living cell. Results and Discussions Design of the Asymmetric Nanopore Electrode

As shown in Figure 1a-b, by coating the interior of an asymmetric nanopore with a conductive Au layer, a certain polarization potential difference is generated between the two terminals of the Au-coated part along the external electric field orientation.17-22 When the polarization potential, ∆Vmin, between the two poles of the ANE is sufficient, a redox equilibrium can be carried out at the two poles of the ANE. Under a negative applied potential (Ebias), the cis opening of the ANE tip serves as the cathodic pole (δ-), whereas the terminal towards the trans opening acts as the anodic pole (δ+). In the absence of a redox reaction, the bare nanopore or unmodified ANE generates a stable current baseline without any signals in the presence of a target species (Figure 1d-e). If a pair of redox reactions coupled with the reduction of H+ occurs at the two poles of an ANE, the production of a hydrogen bubble induces a clear current amplification, which responds to the oxidation at the anode. As a model system, an ANE was modified with a derivative of catechol, 4-thiol catechol (CS), and denoted as CS-ANE. At a sufficient ∆Vmin, the catechol was oxidized to o-


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benzoquinone at the anodic pole of the CS- ANE, whereas the reduction of H+ occurred at the cathodic pole (Figure 1f, left). In the presence of NADH, the oxidized o-benzoquinone electro-catalyzed the oxidation of NADH, thus yielding reduced

catechol. Therefore, the catalytic process leads to the fast generation of H2 at the cathodic pole (Figure 1f, right). This generalized mechanism could be used to

Figure 2 │3D fabrication and characterization of the asymmetric nanopore electrode. (a) Step-by-step fabrication process of the modified ANEs. A gold layer is coated on the interior of the nanopore. (b) Picture of the fabricated ANEs. A 0.1 M HCl solution was used to remove the outer coating.23 (c) Side-view SEM image of an ANE. (d) SEM images of the interior side-view of an FIB-sculpted ANE. The sculpting direction of the ANE is marked with a white arrow. Images of the FIB-sculpted ANE along the z-length from the tip (z = 0 μm) to the trans side (z = – 18 μm), with points marked as I, II and III. The Au layer is clearly shown with high contrast inside the ANE from z = 0 μm to z = – 15 μm. (e) Top-view SEM images of a bare nanopore (top) and an ANE (bottom). (f) I-V responses of the bare nanopore (red), the ANE (yellow) and the catechol-modified ANE (blue). (g) I-V responses of 8 individual catechol-modified ANEs. The I-V curves were measured in a 10 mM PBS solution. (h) Normalized potential drop at the Au layer/solution interface at an applied potential of – 0.6 V. Images 1, 2, 3 from left to right represent three parts of the nanopore at z-lengths of 0 μm, – 2.5 μm and – 4.8 μm, respectively. probe not only NADH but also a wide range of redox species, e.g., dopamine and horse radish peroxidase. The detailed sensing mechanism of the ANE will be discussed in the following section. Fabrication and modification of ANEs A nanopore with a diameter of ~110 nm was coated with an Au layer through electron-beam evaporation. (Figure 2a-c, Supporting Experimental Methods and Figure S1). Then, a 0.1 M HCl solution was used to remove the Au coating from the exterior of the ANE under a continuous flow of N2 through the ANE. Therefore, an Au layer was present only inside the nanopore. The hollow ANE allowed for mass transport through its lumen. To demonstrate the existence of Au layer inside the ANE, we sculpted the sidewall of the ANE along its z length by using a focused ion beam (FIB) and then per-

formed SEM characterization. The SEM images and energy dispersive spectroscopy (EDS) results shown in Figures 2d and S2 indicate that there are approximately 15 μm continuous and uniform Au layers away from the cis opening of the ANE, which is in contrast to the clean interior of the bare nanopore (Figure S3). The SEM image from the top view shows that the diameter of the ANE is 90 nm with an ~ 10 nm Au layer inside (Figure 2e). Due to the negative charge in the inner wall of glass nanopore, the current–voltage (I–V) curve of the bare nanopore shows current rectification (Figure 2f).24-25 After coating with a gold layer, the rectification ratio, R, decrease to 1.3 compared with that of the bare nanopore (R = 4.4), indicating a reduction in the surface charge on the inner wall of the nanopore26-27. Here, we define the rectification ratio, R, as the ratio of the negative current value, I-, to the positive cur-



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rent value, I+, at ± 1.0 V. More importantly, the I-V curve of the ANE remained constant for >5 h (Figure S4). This result reveals the good stability of the gold layer inside the ANE during our entire experiment.

CS-ANEs exhibit good noise performance, which ensures the ability of the CS-ANEs to record the relatively small ionic responses induced by redox reactions (Figure S7).

In the next step, the ANE was functionalized with a derivative of catechol in which catechol was employed as the electrochemically reactive unit and thiol was acted as the anchoring unit for binding to the gold surface (Figures 1a and 2a). The IV curve of the catechol-modified ANE (CS-ANE) reveals that the successful chemisorption of the catechol derivative significantly increased the current values at negative potentials, inducing an increase in the degree of rectification (Figure 2f). A negatively charged surface resulted from the exposure of the hydroxyl groups of the catechol-derivative-modified ANE, leading to a larger R. This result is consistent with the previous study of glass nanopores.28 The R value from > 50 CS-ANEs was calculated to be 12.3 ± 2.5, which shows that our simple fabrication process easily permitted the reproducible fabrication of batches of ANEs with uniform electrochemical performances (Figures 2g and S5). In addition, the successful introduction of catechol on the ANEs was confirmed by both XPS and TOF-SIMS measurements (Figure S6). Importantly, the spectrum of the power spectral density demonstrated that the

Hydrogen-Bubble-Induced Current Amplification The distribution of the electric potential along the ANE depends on the geometry of the conical nanopore. Therefore, to determine the potential drop in the ANE, we performed finite element simulation (FEM) to simulate the asymmetric potential distribution in the ANE based on the coupled PoissonNernst−Planck (PNP) and Navier−Stokes (NS) equations (see Supporting Information for details, Figures S8-S11 and Tables S1-S2). The geometry of the model ANE was based on the SEM image of the ANE, which is rANE = 45 nm, θ = 5° and lAu film = 15 μm (Figures 2d and S1). The surface charge density of the polarized Au layer in the pore is described by a logistictype function which assumes a junction between two oppositely charged parts in the ANE. The peak of the surface charge density sets to - 0.01 C/m2.29 The simulated I-V response and the potential distribution along the ANE are shown in Figure S12.

Figure 3 │ Real-time monitoring of the redox target by using the asymmetric nanopore electrode. (a) Enhanced current modulations of the catechol-modified ANE without (top) and with 0.1 nM NADH (bottom). The ion current time series of the catechol-modified ANE were recorded at Ebias values ranging from – 0.6 V to – 0.9 V. (b) Current histograms of the event oscillations in the absence (green) and presence (blue) of NADH at an applied potential of – 0.6 V. Here, I is the current amplitude of the event, I0 is the open state of the ANE, and ΔI/I0 is the current modulation of the event (Figure S13). Statistical event frequencies


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for the catechol-modified ANE in the absence (c) and presence of NADH (d). (e) Illustration of nanobubble generation (i-ii) followed by dissolution (iii-iv) in the absence (top) and presence (bottom) of NADH and the corresponding signals. More characteristic events are shown in Figure S13. The arrow on the surface of the bubble represent the flux of H2 into/out of solution surface. The blue color represents the proposed distribution of concentration of H+ around the nanobubble and electrode. The color plot from deep to light corresponds to the decrease of H+ concentration. (f) Simulation of the current density-radius trace for a nanobubble at the surface of the cathodic pole at a – 0.6 V applied potential in a 10 mM PBS solution. Details of the FEM simulation can be found in the Supporting Information. The results revealed that the potential difference (ΔVa-c) between the two terminals of the 15 μm ANE was close to the applied external potential of Ebias, which would facilitate easy control of the redox reaction at the ANE. Furthermore, the simulated potential drop at the Au layer/solution interface demonstrates that the applied external potential induced the polarization of the ANE (Figure 2h). For the catechol-modified ANE under these conditions, the catechol is oxidized to obenzoquinone at the anodic pole, whereas the reduction of H+ occurs at the cathodic pole, as follows: 𝐶𝑎𝑡𝑒𝑐ℎ𝑜𝑙 → 2𝐻 ! + 2𝑒 ! + 𝑜-𝐵𝑒𝑛𝑧𝑜𝑞𝑢𝑖𝑛𝑜𝑛𝑒 𝐸 = 400 mV versus SHE

(1)

2𝐻 ! + 2𝑒 ! → 𝐻! 𝐸 = 0 mV versus SHE

(2)

The potential difference between the anodic reaction and the cathodic reaction shows that the redox reaction could occur with an applied potential higher than ∆Vmin of 0.4 V. As expected, pulse-like signals occurred under a negative potential from – 0.6 V (Figure 3a-b). The all-point histogram in Figure S13 for the baseline of the CS-ANE at – 0.6 V shows a good signal-to-noise ratio for the detection of current oscillations. The control experiments performed with the unmodified ANE in 10 mM PBS showed that no signals were obtained at a negative applied potential (Figures 1e and S14). These results confirm that the enhanced pulse-like signal comes from the redox reactions occurring at the CS-ANE. Moreover, there were no current oscillations when both the internal and external solutions were an aprotic acetonitrile solution containing 10 mM tetrabutylammonium hexafluorophosphate (Figure S15). This result further illustrates that the current oscillations originate from the proton reduction driven by the bipolar electrochemistry. Additionally, previous studies have reported that the generation of hydrogen nanobubbles at the surface of a microor nanoelectrode induces the occurrence of pulse-like signals.30-31 Bipolar electrochemical research has demonstrated that the reduction of H+ and the oxidization of hydroquinone occur at the cathode and anode surfaces, respectively, of the conductive object.32 Because the cathodic pole of the ANE is located at the most sensitive constriction of the nanopore, it could be expected that the hydrogen nanobubbles formed at the surface of the cathode would generate distinguishable current oscillations. Since the reduction of H+ leads to the absorption of OH- at the gas/water interface of the nanobubbles,33-34 the hydrogen nanobubbles caused significant negative charges to accumulate at the tip of the ANE. As a consequence, the conductance of ANE was enhanced (step i-ii, Figure 3e), which is in accordance with the results of previously published nanopore studies of charged nanoparticles.35-36 This mechanism is also supported by the examination of the CS-ANE in a basic PBS solution (pH = 9.5). Since the generation rate of nanobubbles at the reduced H+ concentration of the basic solution decreased significantly, the current traces did not exhibit any oscillations (Figure S16). To further explore the hydrogen-bubble-induced current amplification, we performed FEM simulations based on the

2D non-axisymmetric geometry of the ANE (Figures S9 and S10 and Table S2). In the simulation model, a nanobubble was located at the inner Au surface of the ANE tip. The surface charges of the nanobubbles were set at − 0.004 C/m2.33 Figure 3f shows that the current density dramatically increased from r = 1 nm to 11 nm and reached a constant value at r > 8 nm. The values of current density obtained from the line at z = 0 of the cross section at the ANE tip (Figure S9). The results revealed that the nanobubble-enhanced ion accumulation could overcome the electrolyte volume exclusion effect. Moreover, we simulated the ionic current by 2D axisymmetric geometry with nanobubble along the axis. The result is consistent with that simulated in 2D non-axisymmetric model, revealing the suitability of our model. (Figure S17). The Note that the bubble-induced current enhancement is more pronounced in the low salt concentration of 10 mM PBS (Figure S18). The formation of nanobubbles relies on the dynamic equilibrium of the inward and outward flux of H2 at the cathode, where the former is supported by the reduction of H+ at the cathode of the CS-ANE. To explain the periodic manner of the ionic signals, we suggest that the mechanism of nanobubble dissolution is electrical in character. When the tip of the CS-ANE is plugged with a nanobubble, a high potential drop occurs on the nanobubble, causing a complementary decrease in the in the polarized potential difference (ΔVa-c) between the two terminals of the ANE (Figure S19). This effect promotes a decrease in the supply of the inward flux of H2, leading to nanobubble dissolution. Similar to the previously observed nanoprecipitation-assisted ion current oscillations of nanopores,37-38 the formation of nanobubbles causes the local concentration of H+ next to the cathode to be reduced because (1) the nanobubbles hinder the ability of H+ to approach the interface of the Au layer at the cathode where H2 is electrochemically generated; and (2) H+ is driven to the anode by the electric field. Additionally, according to a previous study39, the strong flux of H2 out of the nanobubbles increases with decreasing exposed reactive region at the cathode during bubble growth. Therefore, we suggest that the above effects work together to promote the dissolution of the nanobubbles, leading to the decrease of current to the baseline value (steps ii-iv, Figure 3e). Then, the nanopore is open, and ions can move freely, while the electrochemical reduction of H+ starts to periodically reform a nanobubble. This whole process could produce periodic oscillations in the ionic current. Moreover, the lifetime of the nanobubble in the ANE system is similar to that in previous electrochemical studies (Figure S20).39-40 Figure 3c shows that the event frequency is proportional to the negative applied potential. As the applied potential decreased from – 0.6 V to – 0.9 V, the event frequency, f, increased from 10 s-1 to 71 s-1 (Figure 3c). It has been demonstrated that higher concentrations of hydrogen molecules facilitate the formation of nanobubbles.41 Therefore, a more negative applied potential may accelerate the generation of H2, thus giving rise to an increased event frequency. Since the oxidation of catechol at the anode is coupled with the reduction of H+ at the cathodic



pole, the event frequency could be linked to the amount of oxidized catechol. Real-time Monitoring of NADH Via the CS-ANE As the redox reaction could be monitored in real time by recording the ionic current, we further added the redox target NADH into the external solution (Figure 3a-b). In the presence of NADH, the oxidized o-benzoquinone can electrocatalyze the oxidization of NADH.42-43 At the same time, the obenzoquinone molecules are reduced to the catechol form and are thus available for the next cycle, thus generating a redox cycle that can continuously oxidize NADH at the anodic pole of the ANE, as follows: 𝐶𝑎𝑡𝑒𝑐ℎ𝑜𝑙 → 2𝐻 ! + 2𝑒 ! + 𝑜-𝐵𝑒𝑛𝑧𝑜𝑞𝑢𝑖𝑛𝑜𝑛𝑒 𝑜-𝐵𝑒𝑛𝑧𝑜𝑞𝑢𝑖𝑛𝑜𝑛𝑒 + 𝑁𝐴𝐷𝐻 → 𝐶𝑎𝑡𝑒𝑐ℎ𝑜𝑙 + 𝑁𝐴𝐷 !

This catalytic process was confirmed by measuring the cyclic voltammogram of NADH with a thiol-catechol-modified Au microelectrode (Figure S21). For the CS-ANE, the mediated oxidation of NADH at the anode promotes the generation of nanobubbles at the cathode, causing an immediate increase in the event amplitude and

Figure 4 │ NADH detection in a single live cancer cell by the CS-ANE. (a) Event frequency as a function of the NADH concentration. (b) Relative current enhancement amplitude for 10 pM NADH and 0.1 mM of the typical interferents cysteine (Cys), glucose (Glu), and glutathione (GSH) in 10 mM PBS. The green bar corresponds to the relative ∆I/I0 for the CS-ANE without the addition of analyte in 10 mM PBS. The typical current traces are shown in Figure S23. (c) Representative micrographs of a CS-ANE inserted into an MCF-7 cell and after retraction of the ANE. The cells did not show any morphological changes, remaining intact over the course of the insertion and measurement processes and surviving after retraction. (d) Event frequency obtained by penetrat-

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ing the MCF-7 cell three times with the same CS-ANE. (e) Real-time intracellular NADH measurements with the CSANE inserted into normal and Taxol-treated MCF-7 cells at a recording time of 2 min. Additional current traces are shown in Figure S24. (f) Current histograms of the events from the normal MCF-7 cell (blue) and the Taxol-treated MCF-7 cell (red). The inset shows the event frequency for the catecholmodified ANE in the normal (blue) and Taxol-treated MCF-7 cells (red). prolonging the lifetime of the H2 nanobubbles from 0.31 ms to 1.94 ms at – 0.6 V (Figures 3b and S20). The rapid and strong influx of H2 facilitates the fast nucleation of nanobubbles in the next cycle, leading to a high event frequency (Figure 3d). Notably, NADH could not be detected without the modification of the ANE with catechol at negative potentials from – 0.6 V to – 1.0 V (Figure S22). These results confirm the applicability of the developed CS-ANE for the in-situ monitoring of redox targets. The results of experiments performed with different concentrations of NADH on the CS-ANE are shown in Figure 4a. Because the electrons required at the cathodic pole come from a proportional oxidation reaction at the anodic pole, the increase in the number of oxidized catechol molecules induced by an increase in the NADH concentration leads to a higher rate of nanobubble generation.19,22 Therefore, the event frequency increased from 50 s-1 to 111 s-1 at – 0.6 V when the concentration of NADH was increased from 1 pM to 10 nM. The existence of NADH could be unambiguously confirmed at concentrations of ~1 pM and higher. Interference by extraneous compounds create an obstacle in evaluating highperformance electrochemical sensors in cell-probing applications. To further verify the feasibility of the ANE for NADH detection in a single living cell, we performed experiments in the presence of 0.1 mM concentrations of the typical interferents present in cells, including cysteine (Cys), glutathione (GSH) and glucose (Glu). Figure 4b shows the degree of current enhancement obtained in the presence of the various interferents and a blank PBS solution. The interferents cannot convert the o-benzoquinone into the catechol for the next cycle oxidation. They produce the small current enhancement in CS-ANE compared to the NADH. Therefore, the presented CS-ANE exhibits a highly selective response to NADH. Probing NADH in a Living Cell with the ANE With its high sensitivity and selectivity, the CS-ANE is expected to be a powerful tool for probing intracellular NADH. More importantly, the small diameter of the ANE causes minimal defects in single cells, making it suitable for single living cell detection. In this study, we employed the CS-ANE to measure intracellular NADH in a human breast cancer MCF7 cell (Figure 4c-f). In our experiment, the CS-ANE was immersed in a buffer solution and manually approached a cell under an applied potential of – 0.2 V (Figure 4c). The brightfield image of the MCF-7 cell shows that the penetration of the CS-ANE did not induce any morphological changes. The event frequency for three consecutive penetrations of the cell with the same CS-ANE also demonstrates that the insertion and electrochemical measurement processes have minimal effects on the cell function. The CS-ANE exhibited constant current responses during our single-cell measurements. Figure 4e and Figure S24 shows the signals acquired by the CS-ANE in the MCF-7 cell at – 0.6 V, which were recorded at 2 min, 4 min, 6 min and 8 min, respectively. The current amplitude was calculated to be ∆I/I0 = 0.081, which is consistent with the


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in vitro measurement of ∆I/I0 = 0.099 (Figure 4f). The unmodified ANE did not show any signals when penetrating the cell (Figure S25). To further demonstrate the applicability of the CS-ANE for single-cell analysis, the MCF-7 cell was treated with an anticancer drug, Taxol, which could lower the concentration of NADH by suppressing intracellular metabolism.44 Because Taxol induces a decrease in the NADH concentration, it significantly decreased the current amplitude and frequency for the CS-ANE. The current histogram showed that ∆I/I0 = 0.038 in the presence of Taxol. This value is similar to the current response of the CS-ANE in the absence of NADH (∆I/I0 = 0.058, Figure 3b). The lower frequency in Figure 4f clearly revealed the inhibition of the NADH concentration in the Taxol-treated MCF-7 cell. The obtained results demonstrate the superior ability of the CSANE to act as a wireless nanoelectrode for the in vivo detection of electroactive substances in a single living cell. Conclusions In summary, we have fabricated a novel asymmetric nanopore electrode (ANE) with advantages of a simple fabrication technique, easy modification, and controllable dimensions. We for the first time amplified the unmeasurable nanopore current response of nicotinamide adenine nucleotide (NADH) into a clear pulse-like current signal, facilitating the sensitive monitoring of NADH concentrations as low as 1 pM. Furthermore, our studies demonstrated the high potential of the CS-ANE for application in the in vivo probing of the effect of an anticancer drug (Taxol) on a single living cell. Since the diameter of the ANE could be controlled to as small as 30 nm, the ANE can be precisely inserted into cellular compartments (e.g., the nucleus, mitochondria and lysosomes) to record intracellular electrochemical processes and monitor the interactions with the redox-active molecules. By modifying with a broad range of redox probes, the ANE offers wide applications in redox metabolism characterization, including probing peroxiredoxin, glutaredoxin, neurotransmitters and catalase. Therefore, the ANE can be used as a general electrochemical methodology for characterizing many types of redox-active species in live cells. Note that the bubble-induced current amplification could be extended from the reductiongenerated H2 nanobubbles to oxidization-induced O2 nanobubbles45, which ensures the wide feasibility of this technique. Furthermore, the wireless property of the ANE makes it easier to detect different targets simultaneously by the use of a theta nanopore or nanopore arrays. Benefitting from the superiority of this novel ANE, it is possible to achieve a further understanding of the effects of drugs on cellular redox metabolism and the critical cellular processes and to monitor the cellular and cell-to-cell communications. Consequently, the ANEs hold great significance in analyzing important bodily processes in multicellular and unicellular organisms. Methods The 110 nm glass nanopore was fabricated by using a P2000 capillary puller. The pulling of pipettes followed a twostep process (Line 1: Heat 350, Fil 3, Vel 30, Del 220, Pul 0, Line 2: Heat 350, Fil 2, Vel 27, Del 180, Pul 250). The pulled nanopores were coated with a gold layer using electron beam evaporation (Denton) with a deposition speed of 1 Å/s. SEM characterization and FIB sculpting were performed by using a Zeiss Ultra Plus scanning electron microscope and a Zeiss focused ion beam/field-emission scanning electron microscope dual-beam system (Carl Zeiss, Oberkochen, Germany). The

ANEs were sputtered with Au for 20 s before SEM characterization. Then, a 0.1 M HCl solution was used to remove the Au coating from the exterior of the ANE under a continuous flow of N2 through the ANE. The synthesis of thiol-catechol was reported in previous research.42 The ANE was immersed in a 1 mM solution of catechol in degassed absolute ethanol for 12 h and then washed with absolute ethanol. Finally, the CSANE was dried with N2 before the electrical measurements. The electrochemical measurements were performed in a 10 mM PBS solution with an Axonpatch 700B low-noise amplifier. The internal low-pass Bessel filter of the amplifier was set to 5 kHz. The recorded current was sampled at 100 kHz. The complete experimental details are given in the Supporting Information.

ASSOCIATED CONTENT Supporting Information The Supporting Information including the detailed materials and method, characterization of ANE and CS-ANE, description of simulations, control experiments for supporting the potential mechanism for CS-ANEs, control experiments of NADH detection is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author [email protected]

Author Contributions Y.-L. Y. and Y.-X. H. contribute equally to this work.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (21421004 and 21505043), Innovation Program of Shanghai Municipal Education Commission (2017-0107-00-02-E00023), the “Chen Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation（17CG27）and the Fundamental Research Funds for the Central Universities (222201718001, 222201717003, and 222201714012).

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SYNOPSIS TOC The illustration of asymmetric nanopore electrode for NADH probing in a single living cell.



Figure 1 │ Asymmetric nanopore electrode for monitoring electron transfer dynamics in live cells. (a) Illustration of an asymmetric nanopore electrode (ANE) for probing redox-active species. The applied bias potential (Ebias) drives the potential difference at the two terminals of the Au-coated asymmetric nanopore. At a negative potential, the tip opening (cis) is polarized as a cathode (δ-), while the opposite terminal (trans) acts an anode (δ+). The gradation of the Au layer from red to yellow represents the polarization. Reduction occurs at the cathode, while oxidation occurs at the anode of the ANE. (b) The ANE for single-cell probing. The intracellular redox specie (e.g., NADH) diffuses into the cis tip of the ANE through the tip opening; thus, a pair of redox reactions occur at the cath-ode (δ-) and anode (δ+). The ANE acquires a faradaic current response at the interface of the Au layer through a distinguishable transient ionic current response pattern with a high current and high temporal resolution. (c) A traditional nanoelectrode (e.g., carbon nanoelec-trode) for probing intracellular redox species. The redox reaction occurs at the interface of the solid electrode wire tip, generating a cyclic voltammogram with a low signal-to-noise ratio and poor temporal resolution. (d) The bare nanopore does not produce any transient responses to NADH. (e) The unmodified ANE generates a stable baseline without any signals. (f) The 4-thiol-catechol-modified ANE (CSANE) generates an enhanced current signal due to the generation of H2 nanobubbles at the cathodic pole. Continuous current modulations were recorded at -0.7 V. Left: the catechol can be oxidized electrochemically to the corresponding o-benzoquinone at the anodic pole, whereas H2 is generated at the cathodic pole. Right: in the presence of NADH, the o-benzoquinone/catechol conversion is mediated by the redox couple NADH/NAD+, leading to an increased current response with a high amplitude. Ebias is set at 0.7 V. The electrolyte solution is 10 mM PBS. A pair of Ag/AgCl electrodes is used to apply the bias potential. The electrode close to the tip of the nanopore is defined as the virtual ground. 156x91mm (300 x 300 DPI)


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Figure 2 │3D fabrication and characterization of the asymmetric nanopore electrode. (a) Step-by-step fabrication process of the modified ANEs. A gold layer is coated on the interior of the nanopore. (b) Picture of the fabricated ANEs. A 0.1 M HCl solution was used to remove the outer coating.23 (c) Side-view SEM image of an ANE. (d) SEM images of the interior side-view of an FIB-sculpted ANE. The sculpting direction of the ANE is marked with a white arrow. Images of the FIB-sculpted ANE along the z-length from the tip (z = 0 µm) to the trans side (z = - 18 µm), with points marked as I, II and III. The Au layer is clearly shown with high contrast inside the ANE from z = 0 µm to z = - 15 µm. (e) Top-view SEM images of a bare nanopore (top) and an ANE (bottom). (f) I-V responses of the bare nanopore (red), the ANE (yellow) and the catecholmodified ANE (blue). (g) I-V responses of 8 individual catechol-modified ANEs. The I-V curves were measured in a 10 mM PBS solution. (h) Normalized potential drop at the Au layer/solution interface at an applied potential of - 0.6 V. Images 1, 2, 3 from left to right represent three parts of the nanopore at zlengths of 0 µm, - 2.5 µm and - 4.8 µm, respectively. 164x123mm (300 x 300 DPI)



igure 3 │ Real-time monitoring of the redox target by using the asymmetric nanopore electrode. (a) Enhanced current modu-lations of the catechol-modified ANE without (top) and with 0.1 nM NADH (bottom). The ion current time series of the catechol-modified ANE were recorded at Ebias values ranging from - 0.6 V to - 0.9 V. (b) Current histograms of the event oscillations in the ab-sence (green) and presence (blue) of NADH at an applied potential of - 0.6 V. Here, I is the current amplitude of the event, I0 is the open state of the ANE, and ∆I/I0 is the current modulation of the event (Figure S13). Statistical event frequencies for the catechol-modified ANE in the absence (c) and presence of NADH (d). (e) Illustration of nanobubble generation (i-ii) followed by dissolution (iii-iv) in the absence (top) and presence (bottom) of NADH and the corresponding signals. More characteristic events are shown in Figure S13. The arrow on the surface of the bubble represent the flux of H2 into/out of solution surface. The blue color represents the proposed distribution of concentration of H+ around the nanobubble and electrode. The color plot from deep to light corresponds to the decrease of H+ concentration. (f) Simulation of the current density-radius trace for a nanobubble at the surface of the cathodic pole at a - 0.6 V applied potential in a 10 mM PBS solution. Details of the FEM simulation can be found in the Supporting Information. 163x131mm (300 x 300 DPI)


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Figure 4 │ NADH detection in a single live cancer cell by the CS-ANE. (a) Event frequency as a function of the NADH concentration. (b) Relative current enhancement amplitude for 10 pM NADH and 0.1 mM of the typical interferents cysteine (Cys), glucose (Glu), and glutathione (GSH) in 10 mM PBS. The green bar corresponds to the relative ∆I/I0 for the CS-ANE without the addition of analyte in 10 mM PBS. The typical cur-rent traces are shown in Figure S22. (c) Representative micro-graphs of a CS-ANE inserted into an MCF7 cell and after re-traction of the ANE. The cells did not show any morphological changes, remaining intact over the course of the insertion and measurement processes and surviving after retraction. (d) Event frequency obtained by penetrating the MCF-7 cell three times with the same CS-ANE. (e) Real-time intracellular NADH measurements with the CS-ANE inserted into normal and Tax-ol-treated MCF-7 cells at a recording time of 2 min. Additional current traces are shown in Figure S23. (f) Current histograms of the events from the normal MCF-7 cell (blue) and the Taxol-treated MCF-7 cell (red). The inset shows the event frequency for the catechol-modified ANE in the normal (blue) and Taxol-treated MCF-7 cells (red).



83x106mm (300 x 300 DPI)


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Asymmetric Nanopore Electrode-Based Amplification for Electron

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