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Fabrication of flexible and stretchable nanostructured gold electrode using a facile ultraviolet irradiation approach for nitric oxide detection released from cells Xu Zhao, Keqing Wang, Bo Li, Chao Wang, Yongqi Ding, Changqing Li, Lanqun Mao, and Yuqing Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01088 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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Analytical Chemistry

Fabrication of flexible and stretchable nanostructured gold electrode using a facile ultraviolet irradiation approach for nitric oxide detection released from cells

Xu Zhao,† Keqing Wang,† Bo Li,† Chao Wang,† Yongqi Ding,† Changqing Li,† Lanqun Mao,‡ Yuqing Lin*, † †Department of Chemistry, Capital Normal University, Beijing 100048, China ‡Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences (CAS), Beijing 100190, China

*Corresponding author Tel.: +86 1068903047; Fax: +86 1068903047 E-mail address: [email protected] (Y. Lin). 1 / 18

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: We develop a simple and environmentally friendly ultraviolet (UV)

irradiation-assistant technique to fabricate stretchable nanostructured gold film as a flexible electrode for detection of NO release. The flexible gold film endows the electrode with desirable electrochemitry stability against mechanical deformation including bending to different curvatures and bearing repeated bending circumstances (200 times). The flexible nanostructured gold electrodes can catalyze NO oxidation at 0.85 V (as opposed to Ag/AgCl) and detect NO within a wide linearity in the range of 10 nM-1.295 μM. The excellent NO sensing ability, stretchability together with the biocompatibility allows the electrode for the electrochemical monitoring of NO release from mechanically sensitive HUVECs both in their stretching-free and stretching states. This result paves an effective and easily accessible platform to design stretchable and flexbile electrodes and opens more opportunities for sensing of chemical signal molecules release from cells or other biological samples during mechanical stimulation.

Keywords: Nanostructured gold film; UV irradiation; Flexible electrode; NO; Cell

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INTRODUCTION Flexible and stretchable tactile sensors have received increasing attraction for their facile conformal integration into transformable systems and ability to adapt to different forms and surfaces.1 Despite advances in such sophisticated products, it still remained one of the most challenges to reasonably design microstructured and nanostructured organic1-3 and inorganic conductive electronic materials intimately integrated with stretchable and elastomeric substrates 4. In particular, the reliability and function of the flexible sensors often relies on the stretchable conductive compartment across the whole device.5,

6

graphene,

9, 10

7, 8

carbon nanotubes (CNTs),

conductive polymers,

15

Thus, various nanomaterials such as metal nanowires (NWs),

11-14

and

have been developed to fabricate conductive parts as

stretchable and transparent electrodes. However, these CNT/graphene-based stretchable electrodes always suffer from low electrical conductivity originating from the inherent defects in materials or the fabrication process.9 Thus, metal NW networks have attracted much attention owing to their excellently higher conductivity and mechanical flexibility.16,17 For instance, Pyo presented a reliable method to develop wavy configurations in Ag NW networks with enhanced electromechanical stability as compliant electrodes.18 Lee et al. introduced an Ag-Au core-shell (AACS) nanowire network as wearable energy transfer application.14 Recently, Moon reported an Ag/Au/polypyrrole core-shell nanowire network for stretchable, flexible, and transparent supercapacitors.19 Cheng’s group developed simple yet efficient, low cost approach to fabricate ultrathin AuNWs for strain sensing and supercapacitor.13,20,21 Huang’s group designed Au nanotubes (NTs) on polydimethylsiloxane (PDMS) as a powerful flexible electrochemical sensor aiming to monitor signals from mechanotransduction process.22 Despite the great advantages of metal NWs for preparation of stretchable electrodes, there are fewer reports of using metal nanoparticles for facile construction of stretchable electrochemical sensors except for Someya et al. reported a printable elastic conductor containing Ag nanoparticles.23 In this paper, we report a facile and simple ultraviolet (UV) irradiation-assistant 3 / 18



technique for developing a flexible electrode by in situ growth of a nanostructured gold film on an elastomeric and transparent PDMS substrate for monitoring of NO release from cells.24,

25

As schematically described in Scheme 1, the fabrication

process of the nanostructured Au/PDMS film involved of two consecutive UV irradiation steps. This two-step wet deposition approach to fabricate flexible electrodes is simple, time-saving and environmentally friendly. The obtained flexible electrodes display satisfactory electrical and mechanical properties against mechanical deformation. The flexible nanostructured Au/PDMS film electrode could record NO release both under normal and mechanical strain states from human umbilical vein endothelial cells (HUVECs).

Scheme 1. Schematic representation (not to scale) of the fabrication processes for nanostructured Au/PDMS film flexible electrode. EXPERIMENTAL SECTION

Fabrication of Flexible Nanostructured Au/PDMS Film Electrode. PDMS film (~300 μm thick) was prepared by the spin-coating method. The liquid prepolymer and cross-linker (w/w=10:1) were spin-coated on a glass slide and annealed at 80°C for 1 h. PDMS film was then exposed to UV light at a lamp-to-film distance of 2 cm for 3 h. Furthermore, 8 mM chloroauric acid and 95% ethanol was mixed with a volume ratio of 3:2 to form a gold-plating solution. Next, PDMS film was placed in the 4 / 18


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gold-plating solution and exposed to UV light at the same distance for 3 h to form golden film. Finally, the nanostructured Au/PDMS film was peeled off and contacted with a copper wire via carbon paste. The entire procedure is schematically illustrated in Scheme 1.

RESULTS AND DISCUSSION

Fabrication of Nanostructured Au/PDMS Film Electrode. The rationale for applying the UV irradiation approach for the fabrication of flexible nanostructured Au/PDMS film electrode is to ensure obtain uniform and reproducible film and facilitate the experimental process. The exposure of UV light on PDMS film effectively generate carboxyl groups on the irradiated region, which act as the scaffolding for further surface modifications and thus immobilization of metal particles.

26-29

In addition, previous studies have reported many strategies to create

gold film utilizing UV irradiation synthesis with methoxypolyethylene glycol 30 and NaBH431 etc. as a reducing agent. Here, for the first time, we present a novel synthesis strategy exploiting UV irradiation to prepare gold film, using non-toxic and commonly accessible ethanol as the reductant. In this process, ethanol may produce free radicals and supply the required reducing agent, which facilitates the nanostructured gold film formation. In our work, PDMS film was placed in the gold-plating solution under the UV irradiation with gentle shaking. The solution turns to pink after 1 h irradiation indicating the production of nano-gold particles. After prolonged irradiation (2 h), the formation of yellow gold films on the surface was observed and the bulk of the solution became colorless. However, under the non-UV-irradiated condition, there was no gold film observed at all. On one hand, UV energy exerting on the solution induce excitation and ionization of the solvent and enable the homogeneous distribution of photolytic radicals throughout the whole solution. On the other hand, during the synthesis, UV irradiation also induce the ethanol producing free radicals which provide the necessary reducing agent reacting with the ionic precursor and form gold particles.30 5 / 18



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SEM and Element Characterization of Nanostructured Au Film. In order to verify

that the nanostructured Au/PDMS film was successfully deposited, the morphology, chemical nature, and components of the nanostructured Au/PDMS film were characterized

by

optical

imaging,

scanning

electron

microscopy

(SEM),

energy-dispersive X-ray (EDX) spectroscopy, and elemental mapping. Figure 1a shows an optical image of a nanostructured Au/PDMS film and Figure 1b shows a SEM image. The results distinctly indicate that gold film was homogeneously deposited on the PDMS substrate and possessed an agglomerated nanostructure with a size of approximately 200-400 nm. In addition, elemental Au, Si, and O are revealed in Figure 1c; furthermore, Figures 1d, 1e, and 1f corresponded to elemental mapping of Au, Si, and O, respectively. Therefore, the formation of gold film and the presence of PDMS as the supporting structure are identified, again illustrating that gold film was successfully deposited on the PDMS.

Figure 1. (a) Optical image, (b) SEM image, and (c) EDX analysis of Au film. Corresponding elemental mapping images of (d) Au, (e) Si, and (f) O.

Electrochemical

Characterization

and

Bending

Flexibility

Test

of

Nanostructured Au/PDMS Film Electrode. In this research, the well-prepared nanostructured Au/PDMS film electrode was activated by cyclic voltammetry in a 0.5 6 / 18


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M H2SO4 (Figure 2a), exhibiting characteristic voltammograms for Au redox processes in which the representative reduction peak of the Au electrode appears at approximately 0.9 V. Upon integrating the reduction peak of the Au electrode at 0.9 V in the typical voltammogram in Figure 2a, the charge passed was determined to be 5.252×10-4 C, corresponding to a total active gold surface area of 1.361 cm2.

32

In

addition, the geometry of the electrode area is 0.8 cm2 (length is 1.0 cm, width is 0.8 cm), which is smaller than the active gold surface area, indicating that the electrode surface consists of gold nanoparticles and that the surface is relatively coarse. The larger reaction surface area should be basically attributable to the carboxyl group of PDMS film benefiting the growth of uniformly distributed AuNPs on the surface. Furthermore, no discernible changes in the CV curves were observed, demonstrating its high stability, which can be ascribed to the high adhesion capacity of carboxyl functionalities inducing the Au film to be tightly compacted on the PDMS substrate.

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Figure 2. (a) Voltammetric responses of nanostructured Au/PDMS film electrode in 0.5 M H2SO4 at a scan rate of 100 mV/s. Voltammetric responses of nanostructured Au/PDMS film electrode in 1 mM FcCH2OH solution with the film (b) bent to different curvatures for 10 times, (c) bent for different times with a bending radius of 0.5 cm and Au film outside, and (d) bent for different numbers of times with a bending radius of 0.5 cm and Au film inside.

To test the flexibility and electrochemical stability of the nanostructured Au/PDMS film electrode, CV curves of nanostructured Au/PDMS film electrode (1.0 cm×0.8 cm) were recorded in FcCH2OH subjected to severe conditions, including being circularly bent to different curvatures and for different numbers of times. In Figure 2b, the substrate was bent to different bending radius (from 0.25 to 1 cm) by wrapping on cylindrical objects as shown in the insert. Figure 2b displays a modest bendability under various bending curvatures, manifesting excellent bending tolerance. In Figure 2c, the electrode was circularly bent to a radius of 0.5 cm under different numbers of times (from 0 to 200 times) when the Au film was outside compared to it being inside (Figure 2d). However, the potential and peak currents of FcCH2OH show high repeatability in Figures 2b-2d, evidencing the electrochemical stability of the film against large and repeated mechanical bending. In fact, films of gold formed directly onto PDMS in the microstructured or nanostructured forms spontaneously provide electrodes that can bear large applied strains without fracture since stretchability, i.e. subsequent deformation in this case derives from the motion of inter cracks across the films which form during the fabrication process.33, 34 Electrooxidation and Amperometric Detection of NO on Nanostructured Au/PDMS Film Electrodes. It is of great significance to employ nanostructured Au/PDMS film electrodes for real-time monitoring of NO from mechanically sensitive cells by electrochemical measurements.

35-39

To investigate the oxidation

process of NO on the electrode surface, the CV curve of a nanostructured Au/PDMS film electrode in 0.5 mM NO solution revealed a clear oxidation peak at 0.85 V 8 / 18


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(Figure 3a), exhibiting remarkable catalytic activity to NO oxidation. This may be ascribed to the fact that the Au/PDMS film electrode possesses Au nanostructure, which may have an intrinsic electrocatalytic property for NO.40, 41 Amperometric results on the flexible nanostructured gold electrodes show that a response of NO can be clearly observed. More importantly, a distinct increased current was observed evoked even by 10 nM NO (Figure 3b). The current response is in wide range of linearity to NO concentrations of 10 nM-1.295 μM, with a high sensitivity of 6.16 nA nM-1 cm-2 and a detection limit as low as 1 nM (S/N=3), further demonstrating the excellent electrochemical sensing ability. Consequently, we can detect NO concentration at the nanomole level using a nanostructured Au/PDMS film electrode. In addition, linear regions of NO concentration from 5 to 370 nM are also illustrated and compared before and after stretching (Figure S1). The slight deviation may be caused by the change of nanostructure and of the active area of the film (Figure S2), further confirming the excellent sensing ability of Au/PDMS film electrodes used as flexible electrodes. To understand the mechanism of NO on nanostructured Au/PDMS film electrodes, the effect of the scan rate (v) on the oxidation of 0.15 mM NO solution was investigated by CV (Figure 3c). The oxidation peak current (Ip) of NO is linearly proportional

to

the

scan

rate

(v)

from

5

to

100

mV

s-1

(Ip=1.13×10-6×v(mV/s)+8.72×10-5), indicating an adsorption-controlled process toward NO electro-oxidation on the Au/PDMS film electrode surface (Figure 3d). Simultaneously, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were used to further explore the mechanism of electrocatalytic oxidation of NO on a nanostructured Au/PDMS film electrode, in which the presence of oxygen atoms contributes to the oxidation of NO on the surface of the nanostructured Au/PDMS film electrode (Figure S3).

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Figure 3. (a) CVs of nanostructured Au/PDMS film electrode (0.30 cm × 0.30 cm) in de-aerated phosphate buffer solution with the presence (red line) and the absence (black line) of 0.5 mM NO. (b) Amperometric response of nanostructured Au/PDMS film electrode with the increasing concentration of NO at a potential of +0.85 V (as opposed to Ag/AgCl). (c) Voltammetric responses of a nanostructured Au/PDMS film electrode (1.50 cm × 2.00 cm) in 0.15 mM NO at varied scan rate. (d) Plots of the dependence of anodic peak current as a function of scan rate.

Monitoring NO Release from HUVECs by Nanostructured Au/PDMS Film Electrode. As illustrated in Figure 4a, HUVECs were spindle-shaped and arranged neatly in a clean Petri dish. Without any binder and linker, the human umbilical vein endothelial cells (HUVECs) can grow well on the flexible Au nanostructurer. After 12 h culture, HUVECs on the nanostructured Au/PDMS film electrode reach a density of 10 / 18


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~2×106 cells/cm2, remained in a good state and the majority were spindle-shaped (Figure 4b) during growth and proliferation, indicating the excellent biocompatibility of nanostructured Au/PDMS film electrodes.

Figure 4. Microscopic images of HUVECs (a) in a Petri dish and (b) on a nanostructured Au/PDMS film electrode. (c) Monitoring NO release from HUVECs by nanostructured Au/PDMS film electrode. (d) Comparison of NO release monitoring with nanostructured Au /PDMS film electrode under non-stretching (black line) and stretching states (red line).

In this study, the release of NO was evoked by stimulating HUVECs cultured on a nanostructured Au/PDMS film electrode with L-Arg, which can be enzymatically oxidized by nitric oxide synthase (NOS) to produce NO.42 Here, we report the detection of NO released from HUVECs under the stimulation of L-Arg. In the absence of cells, no current signal appeared when 10 mM L-Arg was added into the surface of the nanostructured Au/PDMS film electrode (Figure 4c, blue line). Obviously, when two different concentrations of L-Arg were injected into the nanostructured Au/PDMS film electrode system cultured with HUVECs, as shown in 11 / 18



Figure 4c (black line), the amperometric current increased gradually, which related to the NO release from cells. In contrast, when 0.3 mM N⍵-Nitro-L-arginine methyl ester (L-NAME) and 10 mM L-Arg were injected simultaneously, no apparent signal was detected (Figure 4c, red line) because L-NAME is a known a inhibitor of NOS. 22 These consequences demonstrate that the current signal (Figure 4c, black line) was caused by NO release from HUVECs upon stimulation by L-Arg. In addition, the electrochemical activity of the nanostructured Au/PDMS film electrode can basically be sustained, although, through the process of culturing HUVECs on the electrodes, the electrode surface was not contaminated (Figure S4). To verify the availability of the nanostructured Au/PDMS film electrode to serve as a flexible electrochemical electrode, HUVECs were seeded on the electrode’s surface, and then the electrode was stretched to 50%, as revealed in Figure 4d (red line). When the 10 mM L-Arg was injected into the stretched nanostructured Au/PDMS film electrode system, the amperometric current increased significantly compared to that using the non-stretched cell. Therefore, the nanostructured Au/PDMS film electrode still possesses the ability to detect NO with the administration of stretching, and, at the same time, the electrochemical properties of the nanostructured Au/PDMS film electrode and cell state are well maintained. The test results are consistent with those in other reports; for instance, 30% strain extents were calculated by the change in length between two neighboring HUVECs, and the electrode can detect more NO, 42 indicating the promising potential of this electrode in monitoring NO under more types of mechanical stimuli. CONCLUSIONS

In summary, we designed and developed a novel, facile, and simple technique to fabricate flexible nanostructured Au/PDMS film electrodes by UV irradiation on PDMS for detection of NO release from HUVECs. Compared with the traditional techniques for design flexible gold electrode, the UV-assisted fabrication strategy is much simple, facile, cheaper and environmentally friendly. The characterization demonstrates that the UV irradiation technique is an effective route to improve the 12 / 18


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fabrication of flexible nanostructured Au film. The flexibility of the proposed nanostructured Au/PDMS film electrode possessed highly mechanical compliance and excellent electrochemical performance under severe conditions, including bending to different curvatures and bearing repeated bending circumstances (200 times). This flexible electrode demonstrated excellent electrocatalytic activity to NO oxidation, allowing monitoring of NO directly from cells. Owing to its excellent biocompatibility, easy functionalization of the Au/PDMS film electrode with rational design will expand its application as flexible device to meet the various requirements such as wearable and vivo-implanted electrochemical sensing.

Acknowledgments This work was financially supported by National Natural Science Foundation (21575090), Beijing Municipal Natural Science Foundation (2162009), Scientific Research Project of Beijing Educational Committee (KM201810028008), and Youth Innovative Research Team of Capital Normal University.

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Fabrication of a Flexible and Stretchable ... - ACS Publications

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