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Sensitive electrochemical detection of nitric oxide release from cardiac and cancer cells via a hierarchical nanoporous gold microelectrode Zhonggang Liu, Ashley Nemec-Bakk, Neelam Khaper, and Aicheng Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01430 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 10, 2017

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Analytical Chemistry

Sensitive electrochemical detection of nitric oxide release from cardiac and cancer cells via a hierarchical nanoporous gold microelectrode Zhonggang Liu,a Ashley Nemec-Bakk,b Neelam Khaper,c Aicheng Chena,* a

Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario P7B 5E1, Canada b Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario P7B 5E1, Canada c Northern Ontario School of Medicine, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, P7B 5E1, Canada *Corresponding author. E-mail address: [email protected] (A. Chen). Tel.:+1 807 3438318; fax: +1 807 3467775. ABSTRACT: The importance of nitric oxide (NO) in many biological processes has garnered increasing research interest in the design and development of efficient technologies for the sensitive detection of NO. Here we report on a novel gold microelectrode with a unique threedimensional (3D) hierarchical nanoporous structure for the electrochemical sensing of NO, which was fabricated via a facile electrochemical alloying/dealloying method. Following the treatment, the electrochemically active surface area (ECSA) of the gold microelectrode was significantly increased by 22.9 times. The hierarchical nanoporous gold (HNG) microelectrode exhibited excellent performance for the detection of NO with high stability. Based on DPV and amperometric techniques, the obtained sensitivities were 21.8 µA µM-1 cm-2 and 14.4 µA µM-1 cm-2, with detection limits of 18.1±1.22 nM and 1.38±0.139 nM, respectively. The optimized HNG microelectrode was further utilized to monitor the release of NO from different cells, realizing a significant differential amount of NO generated from the normal and stressed rat cardiac cells as well as from the untreated and treated breast cancer cells. The HNG microelectrode developed in the present study may provide an effective platform in monitoring NO in biological processes and would have a great potential in the medical diagnostics.

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The biological significance of nitric oxide (NO) was initially discovered in the 1980’s, which revealed that it had the capacity to mediate signaling pathways as a cellular messenger, and played an important role in many physiological processes, including vasodilation, blood pressure regulation, neurotransmission, neural signaling, and immune response.1,2 Thus, the accurate detection and quantification of NO might contribute considerably to a deeper understanding of its essential functions in such physiological processes. To date, a great deal of work has been invested in the detection of NO. Electrochemical techniques may have strong potential in this area due to the excellent merits of high sensitivity, rapid response, easy-use, and low cost.3-5 They have also been employed for many industrial, environmental, agricultural, and clinical applications. Since NO has a particularly close relationship to human health and disease, significant efforts have been invested on the development of electrochemical methods for its detection and quantification in biomedical and clinical applications.6-10 The electrochemical detection of NO primarily based on redox reactions has been acknowledged,11 where electrochemical performance is highly dependent on the effectiveness of the interface between the electrode and the electrolyte.12,13 So far, various approaches have been reported on the design of novel interfaces, in which hemeproteins,14-17 porphyrin or phthalocyanine,18,19 noble metal nanoparticles,20-24 carbon nanotubes25,26 and graphene 27-29 have been predominantly employed. Among these, hemoproteins (hemoglobin, myoglobin, cytochrome c) have been intensely studied to fabricate NO sensors due to their catalytic activity in its reduction.16,17 However, the electroactive groups in the various hemoproteins may be embedded deeply in the proteins, leading to a limitation in the direct electron transfer between the hemoproteins and the electrode surface, which affects the effective sensing of NO.30 Notably, gold nanoparticles have attracted great interest due to their advantages of rapid electron transport,

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efficient catalysis, and large surface area, making them attractive and promising in the development of electrochemical sensors.31,32 A nanoporous structure that was directly grown on a gold microwire exhibited several advantages in comparison to a chemically gold nanoparticlemodified electrode. In the present work, an Au microwire electrode was made and employed as the substrate for the direct growth of a 3D hierarchical nanoporous structure by a facile electrochemical alloying and dealloying method, which was accomplished with cyclic voltammetry performed in a benzyl alcohol (BA) electrolyte that contained ZnCl2. Subsequent to the alloying and dealloying treatment, the electrochemically active surface area (ECSA) of the gold microwire electrode was significantly increased. The electrochemical behaviors of gold microwire and hierarchical nanoporous gold (HNG) microelectrode were investigated comparatively for the electrocatalytic oxidation of NO. The developed HNG microelectrode was further employed for the in-situ monitoring of NO release from rat cardiac H9c2 cell line that were treated with an iron solution, and breast cancer MCF-7 cells that were treated with doxorubicin (Dox). It has been reported that iron overload within cardiac cells may cause oxidative stress, apoptosis, and inflammation.33 Doxorubicin is a common anti-neoplastic agent and doxorubicin-induced toxicity can result in high oxidative stress, and apoptosis.34-36 The responses of the rat cardiac and the breast cancer cell line that were exposed to different levels of stress were evaluated using the electrochemical sensor based on the HNG microelectrode.

EXPERIMENTAL SECTION Chemical Reagents. Gold wire (127 µm in diameter, 99.99%) and zinc foil (99.98%) were purchased from Alfa Aesar. L-arginine (L-arg), benzyl alcohol, ZnCl2, NaH2PO4, Na2HPO4, and

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NaCl were suppied by Sigma-Aldrich. The Griess reagent kit was obtained from Biotium Inc. All other analytical reagents were used as received. The phosphate buffer saline (PBS) solution was comprised of 0.1 M NaH2PO4, 0.1 M Na2HPO4 and 0.1 M NaCl. The PBS solution saturated with NO was prepared as previously reported.11 In brief, prior to preparation, the 0.1 M PBS solution was initially purged with high-purity argon gas for 20 min. Subsequently, pure NO gas was bubbled for 30 min to obtain the saturated NO solution, where its concentration at room temperature was reported as 1.8 mM.37 All standard NO solutions were prepared daily with the appropriate dilution of the stock solution. All aqueous solutions were prepared with deionized water (18.2 MΩ cm) purified by a NANO pure® Diamond™ UV ultrapure water purification system. Fabrication of the HNG Microelectrode. To prepare an Au microelectrode, a 10 cm long copper wire was passed into an eight-cm glass tube, whose end was dipped into a conductive gold paste (Heraeus Inc., USA), which acted as conductive adhesive. Afterward, a 10 mm long gold microwire (127 µm in diameter, 99.99%, Alfa Aesar) was carefully passed through the glass tube until the microwire was gently attached to the copper wire. An epoxy resin was employed to wrap the top and end of the glass tube. The prepared electrode was then introduced into an oven at 60ºC for 90 min, followed by being held vertically in the ambient air to cool. The HNG microelectrode was fabricated by an electrochemical alloying/dealloying method,31 which was based on a three-electrode system (Scheme S-1), where the gold microelectrode, Zn foil, and Zn wire as the working electrode, counter electrode, and reference electrode, respectively. The alloying/dealloying of the Au microelectrode was carried out in a mixture of benzyl alcohol and 1.5 M ZnCl2 via cyclic voltammetry in the potential range between -0.70 and +1.80 V (vs. Zn) at the scan rate of 10 mV s-1 and 110 ºC.

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Instrumentation and Electrochemical Experiments. Scanning electron microscope images were recorded using field emission scanning electron microscopy (FE-SEM, Hitachi SU-70). Xray diffraction (XRD) patterns were recorded via a Philips X’Pert Pro X-ray diffractometer with Cu Kα radiation (1.5406 Å). The electrochemical experiments were performed using a CHI 660D computer-controlled potentiostat (CH Instrument Inc., USA) with a standard threeelectrode system. A gold microwire or HNG microelectrode served as a working electrode, whereas an Ag/AgCl (1 M KCl) electrode and platinum coil were used as the reference electrode and counter electrode, respectively. All solutions were deaerated with high-purity argon for 20 min prior to the electrochemical measurements, which were carried out at room temperature (20 ± 2oC). Cell lines and culture conditions. Rat cardiac cells (H9c2 cells; ATCC) and breast cancer (MCF-7 cells; ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Aldrich, St Louis, MO, USA) that was supplemented with 10% fetal bovine serum (FBS) and an antibiotic/antimycotic (100 units mL-1 penicillin G sodium, 100 µg mL-1 streptomycin sulfate, 0.25 µg mL-1 amphotericin B). Cells were grown in T-150 flasks in a humidified atmosphere at 5% CO2 at 37ºC, and were harvested at 80% confluency. In order to investigate the stress response of the cardiac cells, H9c2 cells were treated with a 50 µM and 100 µM iron solution for 1 h, respectively, where the iron solution was prepared by dissolving ammonium iron(III) citrate into supplemented media. To study the response of the cancer cells to doxorubicin, the cancer cells were treated with different concentrations of doxorubicin hydrochloride (10, 20, or 30 µM) for 4 h, and the untreated cells were used as the control. The NO release from the different cells was measured using the HNG microelectrode via chronoamperometry, where the cell samples were

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spiked with a 0.1 M PBS solution and 10 µL of 4 mM L-arg was successively added into the solution.

RESULTS AND DISCUSSION Surface Characterization and Electrochemical Behavior of the HNG Microelectrode. The

morphologies

of

the

HNG

microelectrode

prepared

via

the

electrochemical

alloying/dealloying method are displayed in Figure 1A and 1B. Prior to treatment, the gold microwire possessed a smooth surface as seen from the SEM image (Figure S-1). Following the alloying/dealloying process, the color of the gold microwire was changed from yellow to dark red. As seen in the low-magnification SEM image (Figure 1A), a porous structure was uniformly formed on the surface of gold microwire. Figure 1B displays the high-magnification SEM image, revealing the formation of 3D interconnected networks with pore dimensions of 300 - 500 nm. Figure 1C presents the XRD patterns of the HNG and the smooth gold microwire, where the peaks derived from the brass substrate were marked by asterisks. After the allying/dealloying process, the peak at 64.7º was disappeared while the peak at 44.5º was highly increased accompanying with the appearance of peak at 38.5º. The peaks located at ca. 38.5º, 44.5º, 64.7º and 77.6º can be indexed to (111), (200), (220) and (311) planes of face-center-cubic (fcc) Au (JCPDS No. 01-1172), respectively.38 The average crystallite sizes of gold microwire and HNG were calculated using Debye-Scherrer equation to be ~43.1 and ~70.4 nm, respectively. The results indicated that the gold atoms were rearranged and the (200) and (111) planes in the hierarchical nanoporous structure of gold microelectrode were preferentially oriented after the alloying/dealloying process.

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The dependence of the alloying/dealloying cycles on the morphology of the HNG microelectrodes was investigated, with the corresponding SEM images presented in Figure S-2. Some nanopores were formed on the surface of the microelectrode subjected to the initial tencycle alloying/dealloying treatment as seen in Supplementary Figure S-2A. Because of the pore formation, the area fraction was decreased to 77.1% (Figure S-3), which was analyzed by Image J (National Institutes of Health). As the number of cycles was increased, the porous structure became more notable, where the pore dimensions as well as the overall porosity were increased. A well-defined 3D hierarchical nanoporous structure was formed following 50 cycles (Figure S2E) and its area fraction was estimated to be 60.3%. However, further increase of the number of cycles to 60 resulted in much larger pore dimensions and enhanced porosity (Figure S-2F) with an area fraction of 51.3%. Subsequently, the electrochemical behaviors of the bare gold microwire and HNG microelectrodes treated with different cycles were studied by cyclic voltammetry in a 0.1 M H2SO4 solution (Figure S-4). The oxidation peak of gold microwire can be observed at ca +1.2 V; while the peak at +0.9 V indicated the reduction of gold oxide (Figure S-4A). Figure S-4B depicts the electrochemical behaviors of HNG microelectrodes treated with different cycles, showing the oxidation and reduction peaks of the HNG microelectrode after the 10-cycle treatment were highly increased compared with the gold microwire electrode. As the cycle numbers of alloying/dealloying were increased from 10 to 50, the reduction peak of gold oxide was gradually increased. While as the alloying/dealloying cycles was further increased to 60 cycles, the reduction peak of gold oxide was decreased. The electrochemically active surface area (ECSA) of the HNG microelectrodes prepared under different cycles was estimated by integrating the charge associated with the reduction peak of gold oxide centred at ~+0.90 V.39,40 The roughness factor expressed as the ratio between the ECSA and the geometrical area of

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electrode was calculated, where the geometrical surface area of the gold microwire electrode was 0.848 mm2 as listed in Table S-1. It was found that the HNG microelectrode treated after 50 cycles possessed the highest surface area, which was determined to be 25.0 mm2 by assuming that the reduction of a monolayer of gold oxide was 390 µC cm-2, and the roughness factor was thus calculated to be 29.5, which was consistent with the aforementioned SEM images displayed in Supplementary Figure S-2. All the results indicated that the HNG microelectrode created from the 50-cycle alloying/dealloying possessed a significantly improved surface area and the network structure. Therefore, the HNG microelectrode with 50-cycle alloying/dealloying was selected as the optimum and employed in the subsequent measurements. We investigated the performance of the optimized HNG microelectrode for the electrochemical oxidation of nitric oxide. Figure 2A shows the cyclic voltammograms (CVs) of the HNG microelectrode and gold microwire electrode recorded in a 0.1 M PBS solution (pH 7.2) in the absence and in the presence of 5.0 µM NO at a scan rate of 20 mV s-1. Only a small and broad NO oxidation peak was observed at around +0.84 V on the gold microwire electrode. In the case of the HNG microelectrode, there was an obvious peak for NO oxidation with a much higher peak current, which was approximately 6.23 times higher than that of the gold microwire electrode. Moreover, the peak potential of NO oxidation (+0.75 V) on the HNG microelectrode was shifted to less positive potential with respect to that of the gold microwire electrode. The significant enhancement of the electrocatalytic activity of the HNG microelectrode could be further confirmed by the Tafel plot. As shown in Figure 2B, the obtained slopes for the HNG microelectrode and gold microwire electrode were 59.0 mV dec-1 and 101 mV dec-1, respectively. It is known that Tafel plots may provide the information on the activation energies of specific electrochemical reactions; and the lower slope for the HNG microelectrode indicated lower

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activation energy in comparison to the gold microwire electrode.41 These results demonstrated that the HNG microelectrode facilitated rapid electron transfer at the interface between the electrode and electrolyte, thus leading to the highly improved electrochemical oxidation of NO. Electrochemical Detection of NO with DPV and Amperometric Techniques. Figure 3A presents the DPV curves of NO oxidation on the HNG microelectrode in a 0.1 M PBS solution (pH 7.2). A well-defined NO oxidation peak appeared at +0.724 V, and the peak current density was increased with the increase of the NO concentration. Figure 3B shows the calibration curve of the HNG microelectrode for NO detection with a high sensitivity of 21.8 µA µM-1 cm-2; an experimental linear range of 0.360 µM to 18.0 µM and a very good correlation coefficient of 0.998. The detection limit calculated based on the equation shown in Supporting Information was 18.1±1.22 nM. Figure 3C depicts the hydrodynamic amperometric NO response of the HNG microelectrode, in which NO, with different concentrations from 5.0 nM to 200 µM, was successively added to a 0.1 M PBS solution (pH 7.2). The insert in Figure 3C illustrates the amperometric NO response at the lower concentrations in the range of 5.0 nM and 1.0 µM, showing that the HNG microelectrode exhibited a rapid and sensitive response to the increase of NO concentrations. The response time was determined to be less than 3 s to attain 95% of the steady-state current. The sensitivity was 14.4 µA µM-1 cm-2 with a coefficient factor (R2) of 0.997 (Figure 3D), and the LOD was calculated to be 1.38±0.139 nM. A comparison of the electrochemical performance with the reported electrochemical NO sensors was summarized in Table S-2. The HNG microelectrode developed in the present work exhibited a wide linear range and a much lower detection limit on the detection of NO. The NO analysis using the HNG microelectrode was further compared with the Griess assay, which is one of the most reliable and specific methods to quantify NO. The NO analysis based on the Griess assay was realized via a

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diazotization reaction, in which the UV-Vis absorbance was measured at the wavelength of 548 nm. The UV-Vis spectra and the calibration plot have been displayed in Figure S-5A and B, respectively. A comparison of the NO analysis obtained with the HNG microelectrode and Griess assay is presented in the Supplementary Table S-3, showing that the analytical results obtained using the HNG microelectrode were in good agreement with those obtained from the Griess assay. It is worth noting that the detection limit of the Griess assay is ~0.1 µM and that the amount of NO release from cells is usually below 0.1 µM. It would be difficult to in-situ monitor the NO release from cells. On the other hand, as seen in Figure 3C, the experimental limit of quantitation of the HNG microelectrode method was 5.0 nM, which could effectively monitor the NO release from cells. The aforementioned results revealed the accuracy and high sensitivity of the HNG microelectrode with a low detection limit in the electrochemical detection of NO. Interference, Stability and Reproducibility of the Electrochemical NO Sensor. Since ascorbic acid (AA), L-cysteine (Cys) and uric acid (UA) may co-exist with the released NO in real biological samples and their oxidation peak potentials are close to the NO oxidation, it is necessary to investigate the potential interference on the electrochemical detection of NO from those species. Figure 4 presents the DPV responses of the HNG microelectrode to NO with different concentrations varied from 0.70 to 6.5 µM in the presence of AA, Cys, UA and L-arg, where the concentration of each was 500 µM. Only a small and broad peak was observed in the potential range from -0.05 to 0.45 V, which might be attributed to the electrochemical oxidation of AA, Cys and UA. When NO was added, the distinct peak for NO oxidation appeared at +0.724 V; and the peak current density was increased in a linear fashion with the increase of the NO concentration as shown in the inset of Figure 4. The sensitivity was calculated to be 20.5 µA

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µM-1 cm-2, which was very close to the value determined from Figure 3B, confirming that NO was selectively detected in the presence of AA, Cys, UA and L-arg with over 100 times higher concentration. In addition, some inorganic species such as Cl-, NO3-, Na+, K+, SO42-, Mg2+ and Cu2+ commonly exist in biological samples. We further tested their impact on the detection of NO using DPV; and no interference was observed (Figure S-6). All those results demonstrated the favorable anti-interference capacity of the HNG microelectrode for the NO detection. Repetitive measurements on the electrooxidation of NO with the HNG microelectrode are presented in Figure 5A. These were realized by measuring the current responses to 5.0 µM NO over 30 cycles, and the data shown in Figure 5A was the peak current density measured at +0.724 V (inset in Figure 5A). No apparent change in the current responses was observed with a relative standard deviation (RSD) of 1.10%. Further, the reproducibility of the HNG microelectrodes (E1–E5) fabricated from different batches was evaluated using DPV. As shown in Figure 5B, the voltammetric responses of NO at different microelectrodes were almost identical with a RSD of 1.18%. These results revealed that the HNG microelectrode exhibited good reproducibility and robust stability under repeated measurements. Electrochemical Analysis of NO Release from the Cardiac and Cancer Cells. The developed HNG microelectrode was utilized to monitor the NO release from the rat cardiac and breast cancer cells in-situ, which was measured through amperometric responses at the applied electrode potential of 0.80 V. To investigate the critical role of NO in biological processes, L-arg was employed to activate nitric oxide synthase (NOS) in living cells, which can generate NO.42 Figure 6A displays the amperometric responses to the NO release from the normal and stressed cardiac cells that were pretreated with an iron solution, where the arrows indicate the addition of 4.0 mM L-arg. As shown in Curve a (black line), there was a negligible response when L-arg

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was sequentially added to the PBS solution in the absence of the cells. In contrast, when the cardiac cells were introduced into the solution, there was a strong current response upon the addition of L-arg (red line). Interestingly, in the case of the cardiac cells that were treated with 50 µM (blue line) and 100 µM iron (pink line), the amperometric responses of NO release from the treated cells were higher than that of the untreated cells, and were also increased as the iron concentration was increased from 50 to 100 µM. In comparison with the normal cells, the current responses of the NO that was released from the stressed cells treated with 50 µM and 100 µM iron were increased by 47.9±3.80% and 125±9.90%, in the first injection of L-arg, respectively, indicating a positive correlation between the current of the NO released and the concentration of iron. The concentrations of NO release from the cardiac cells treated with 50 µM and 100 µM iron for the initial injection of L-arg were estimated to be 8.46±2.78 nM and 12.5±3.14 nM, respectively, for which the amounts of NO released were approximately 1.43 and 2.12 times as high as that of normal cardiac cells (~5.90 ± 2.68 nM NO released), revealing that higher concentrations of iron could induce the increased release of NO from the cardiac cells. However, the amperometric responses toward NO release were decreased in the subsequent injection of Larg. In order to explore the change in NO release from the cardiac cells with the stimulation of Larg, we further examined the responses toward NO in the second and third injection of L-arg in comparison with that of the first injection. Figure 6B presents the decreased percentage in the current response of the NO release from the cardiac cells without pretreatment (red column) and pretreated with 50 µM (blue column), or 100 µM (pink column) iron in comparison with the initial value of NO. The results were calculated based on the following equation:

∆I R =

Ii − Io × 100% Io 12


(1)

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where Io and Ii were the current responses of the NO release from the cells following the first and second (or third) injection of L-arg, respectively. In the untreated cells, slight decreases by 3.57% and 11.9% on the responses of NO release were observed in the second and third injection of Larg, respectively. In the case of the treated cardiac cells, it decreased by 15.2% and 26.9% on the responses of NO release from the cells pretreated with 50 µM iron in the second and third injection of L-arg, respectively; while the decreases were 25.3% and 39.5% for the responses of NO from the cells pretreated with 100 µM iron. A double regulatory relationship between iron and NO has been suggested where iron deprivation of cells results in increased NO production;43 conversely iron overload can lead to decrease in NO release as observed in the present study. We further investigated the NO release from the cancer cells using the HNG microelectrode. Figure 7A displays the amperometric responses to the NO release from the untreated and treated MCF-7 breast cancer cells as L-arg was added. For the untreated breast cancer cells, the current response of the NO was about 0.140±0.0370 µA cm-2 following the first injection of L-arg. When the cancer cells were pretreated with 10 and 20 µM Dox, the current responses to NO release were gradually increased to 0.155±0.0380 µA cm-2and 0.232±0.0490 µA cm-2 respectively at the first addition of L-arg. However, when 30 µM Dox was used, the current response was decreased to 0.204±0.0450 µA cm-2. The concentrations of NO release from the cancer cells pretreated with 10 µM and 20 µM Dox were 12.8±3.18 nM and 18.9±4.04 nM, respectively, in which the quantities of NO released were ~1.09 and 1.62 times higher than that of the untreated cancer cells (11.7±3.06 nM). Several studies have reported the role of oxidative stress in doxorubicin induced cardiotoxicity including the upregulation of antioxidant enzymes NOS and subsequent NO release from the cancer cells.34,36,44 When the cancer cells were pretreated with 30 µM Dox, although the NO concentration was decreased to 16.7±3.70 nM, it

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was still ~1.43 times higher than that of the untreated cancer cells. The decrease of the response to NO release from the cancer cells pretreated with 30 µM Dox could be attributed to the Doxinduced apoptosis.45 The decrease on the responses to NO following the injection of L-arg was further examined. The decreased percentage in the current response of the NO release from the cancer cells is shown in Figure 7B, which was calculated using Equation 1 based on the difference between the first and subsequent responses of the NO release from the cancer cells. In the case of the untreated cancer cells, the decreases of 17.0% and 26.4% on the responses to NO were observed in the second and third injection of L-arg, respectively. However, it was found that decreases in NO release correlated with increased Dox concentration. When the cancer cells were pretreated with 30 µM Dox, the responses of NO were severely decreased by 51.1±5.62% and 64.6±7.13% compared with the initial response following the injection of L-arg, respectively. The results suggested that the cytotoxicity of Dox with high concentrations would induce apoptosis, resulting in the decreased release of NO. All these results have shown that the HNG microelectrode can be employed in monitoring the NO release from the cardiac and cancer cells with a high sensitivity and resolution. The considerable difference in the NO that was released from cardiac or breast cancer cells with or without the pretreatment has been detected, which could be attributed to the higher expression of NOS in the cells that were stimulated with iron or doxorubicin, in contrast to those without pretreatment. It is suggested that the higher expression of NOS reflects an important role of NO under such stressed conditions. These results further revealed the important implications of the effective detection of such biomarkers based on the HNG microelectrode.

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CONCLUSIONS In summary, a gold microelectrode with a 3D hierarchical nanoporous structure was fabricated via an electrochemical alloying/dealloying method, which was developed as a promising interface for the electrochemical sensing of NO. The ECSA of the HNG microelectrode was significantly increased by 22.9 times. The HNG microelectrode exhibited excellent performance for the detection of NO with sensitivities of 21.8 µA µM-1 cm-2 and 14.4 µA µM-1 cm-2, as well as detection limits of 18.1±1.22 nM and 1.38±0.139 nM, via DPV and amperometric techniques, respectively. The HNG microelectrode exhibited the favorable anti-interference and high stability. Moreover, the developed HNG microelectrode provided a novel approach to monitor NO that was released from different cells, revealing that a significant differential amount of NO can be generated from the normal and stressed rat cardiac cells as well as from the untreated and treated breast cancer cells. This sensor may serve as a reliable and effective platform for the elucidation of cellular stress responses and medical diagnostics.

ASSOCIATED CONTENT Supporting Information Supporting Information is available free of charge via the Internet at http://pubs.acs.org. A scheme of the electrochemical system (Scheme S-1), SEM images (Figure S-1, and Figure S-2), area fraction analyzed by Image J (Figure S-3), cyclic voltammograms (Figure S-4), UV-vis spectra (Figure S-5), interference study of inorganic ions (Figure S-6), ECSA and roughness factor (Table S-1) of gold and HNG microelectrodes, a comparison with the modified electrodes on the NO detection (Table S-2), a comparison of the NO analysis with the HNG microelectrode and Griess assay (Table S-3).

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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by a Discovery grant from the Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN-2015-06248). A.C. acknowledges NSERC and the Canada Foundation for Innovation (CFI) for the Canada Research Chair Award in Materials and Environmental Chemistry.

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Figure Captions Figure 1. (A & B) SEM images of the hierarchical nanoporous gold (HNG) microwire treated by electrochemical alloying/dealloying in a mixed electrolyte of benzyl alcohol (BA) and ZnCl2. (C) XRD patterns of the gold microwire and HNG microwire. An asterisk indicates the peaks derived from the substrate. Figure 2. (A) CV response to 5.0 µM NO recorded on the gold microwire electrode and the HNG microelectrode in 0.1 M PBS (pH 7.2) at the scan rate of 20 mV s-1. (B) The Tafel plots obtained from cyclic voltammograms. Figure 3. (A) DPV responses and (C) amperometric responses of the HNG microelectrode for the analysis of different concentrations of NO in 0.1 M of PBS (pH 7.2). The inset in (C) is the enlarged amperometric responses of NO in the low concentration range. The applied potential in (C) was 0.80 V. (B & D) The corresponding calibration plots. Each point was expressed as mean ± standard deviation (n = 3). Figure 4. DPV response to NO with different concentrations at the HNG microelectrode in the presence of 500 µM AA, Cys, UA and L-arg. The inset was the NO calibration plot. Each point was expressed as mean ± standard deviation (n = 3). Figure 5. (A) Stability measurement at the HNG microelectrode with repeated analysis of 5.0 µM NO. (B) Reproducibility on the different batches of the prepared HNG microelectrodes (E1E5). Figure 6. (A) Amperometric responses to NO released from cardiac cells with the successive addition of L-arg in a 0.1 M PBS solution (a), containing cultured cells (b), or cultured cells treated with 50 µM (c) or 100 µM iron (d). Applied potential: 0.80 V. (B) The decreased percentage of the responses of NO in comparison with that one obtained after the first injection of L-arg, respectively. Figure 7. (A) Amperometric responses to NO released from breast cancer cells with the successive addition of L-arg in a 0.1 M PBS solution (a), containing cultured cells (b), or cultured cells pretreated with 10 µM (c), 20 µM (d) or 30 µM (e) doxorubicin (Dox), respectively. Applied potential: 0.80 V. (B) The decreased percentage of the responses to NO in (A) in comparison with that one obtained after the first injection of L-arg, respectively. 20


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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Figure 7.

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Table of Contents (TOC)

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