Gold nanoparticles decorated reduced graphene oxide for detecting the presence and cellular release of nitric oxide

Accepted Manuscript Title: Gold Nanoparticles Decorated Reduced Graphene Oxide for Detecting the Presence and Cellular Release of Nitric Oxide Author: Siong Luong Ting Chun Xian Guo Kam Chew Leong Dong-Hwan Kim Chang Ming Li Peng Chen PII: DOI: Reference:

S0013-4686(13)01564-8 http://dx.doi.org/doi:10.1016/j.electacta.2013.08.036 EA 21060

To appear in:

Electrochimica Acta

Received date: Revised date: Accepted date:

12-3-2013 8-7-2013 5-8-2013

Please cite this article as: S.L. Ting, C.X. Guo, K.C. Leong, D.-H. Kim, C.M. Li, P. Chen, Gold Nanoparticles Decorated Reduced Graphene Oxide for Detecting the Presence and Cellular Release of Nitric Oxide, Electrochimica Acta (2013), http://dx.doi.org/10.1016/j.electacta.2013.08.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Gold Nanoparticles Decorated Reduced Graphene Oxide for

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Detecting the Presence and Cellular Release of Nitric Oxide

Chen a,*

School of Chemical and Biomedical Engineering, Nanyang Technological University, 70

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a

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Nanyang Drive, Singapore 637457 b

Institute for Clean Energy & Advanced Materials, Southwest University, Chongqing

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400715, P.R. China

GlobalFoundries Singapore, 60 Woodlands Industrial Park D Street 2, Singapore 738406,

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Singapore

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Siong Luong Ting,a Chun Xian Guo,b Kam Chew Leong,c Dong-Hwan Kim,a Chang Ming Li,a,b,* Peng

* correspondence to [email protected] or [email protected]

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Abstract

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In this work, we report the preparation of a nanocomposite consisting of gold nanoparticles (AuNPs) electrochemically deposited on electrochemically reduced graphene oxide (ERGO),

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and its use for sub-micromolar detection of nitric oxide (NO). ERGO network provides highly conductive pathways for electron conduction and a large surface area for catalyst

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support, while AuNPs act as efficient electrocatalysts towards the oxidation of NO. The synergistic integration of ERGO and AuNP realizes the electrochemical detection of NO with

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high sensitivity (5.38 μA/μM/cm2), low detection limit (133 nM with a S/N = ~5.5), and a

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fast response time (3 s). Furthermore, we demonstrate the use of the AuNP-ERGO hybrid electrode to detect the dynamic release of NO from live human umbilical vein endothelial

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cells (HUVECs).

Keywords: graphene, gold nanoparticles, electrochemical sensor, nitric oxide detection,

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nanocomposite.

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1. Introduction

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Nitric oxide (NO) was first identified as an endothelium-derived relaxing factor (EDRF) in 1970s [1]. Since then, the research on the regulatory roles of NO in biological systems has

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been fervent. The studies have revealed that NO regulates many physiological functions including blood vessel dilation [2], anti-coagulation [3], neurotransmission [4], and anti-

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inflammation [5]. The excess or deficiency of NO can result in various pathological conditions such as tumor angiogenesis [6], atherosclerosis[7], Parkinson’s disease[8] and

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diabetes [9-10]. Therefore, it is of great importance to accurately quantify nitric oxide level for study of cell functions and diagnosis. However, this is challenged by the low

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physiological concentration of NO and its short life time (~ 5 s) due to its rapid conversion to NOx- by oxygen and superoxides present in biofluids.

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Compared to the detection methods based on fluorescence measurement [11-12] and liquid

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chromatography [13], electrochemical detection allows simple, fast, real-time and

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quantitative detection with high sensitivity. Recently, efforts have been made to increase the sensitivity for electrochemical detection of NO by engineering the electrode with functional nanomaterials [14]. Among them, graphene (a monolayer of carbon atoms two-dimensionally arranged in a honeycomb structure) is of particular interest, due to its high conductivity, large surface area, wide electrochemical detection window, and chemical inertness [15]. Gold nanoparticles (AuNPs), which are biocompatible, have been used in conjunction with graphene for sensor applications [16-17]. AuNPs also exhibit excellent catalytic property towards NO oxidation. Therefore, AuNPs have been composited with various nanomaterials for the development of NO sensors [18-23]. However, they have not been employed for physiological measurements and there is still room to simplify the fabrication process and 3

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improve the performance of NO sensors. And in the current developments, hazardous chemical reagents (e.g. hydrazine) are used to reduce graphene oxide (GO) in graphene preparation. In comparison to such chemical reduction, electrochemical reduction of GO is

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faster and more environmental-friendly [24]. In this work, we report a hybrid film of electrochemically reduced graphene oxide (ERGO)

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and gold nanoparticles (AuNPs) simply made by electrophoretic deposition of GO sheets followed by in-situ electrochemical reduction and subsequent in-situ electrochemical growth

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of AuNPs. We have also demonstrate the use of such hybrid film modified electrode for

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sensitive detection of NO and its dynamic release from live cells.

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2. Experimental Section

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2.1. Materials

Sulfuric acid (H2SO4), nitric acid (HNO3), sodium nitrite (NaNO2), potassium

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hydroxide (KOH), potassium chloride (KCl) and acetylcholine were purchased from SigmaAldrich. ). Phosphate buffer solution (PBS) (pH 7.4) was used as the electrolyte solution for all experiments. All solutions were prepared with deionized water purified from Millipore Milli-Q system.

2.2. Equipment

Electrochemical characterizations and measurements were conducted with a CHI660D electrochemical workstation (CH Instruments, China) using a three-electrode system consisting of a platinum counter electrode, a standard silver/silver chloride electrode 4

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(Ag/AgCl) as the reference, and a glassy carbon working electrode (GCE) without or with coating of AuNPs-ERGO thin film. The morphology and X-ray energy dispersive spectrometry of the samples were examined by a field emission scanning microscope (JEOL

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JSM-6700F). In the comparison experiments, the cylindrical glassy carbon (~5 mm thick) was carefully removed from the commercial electrode (CHI). Epoxy was used to attach a

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copper wire and insulate the glassy carbon with top surface exposed. SEM was then taken before and after electrochemical deposition of AuNPs. UV-vis characterization was

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performed using a UV-vis spectrophotometer (Shimadzu UV-2400PC).

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2.3. Preparation of nitric oxide solution

PBS with saturated nitric oxide was prepared as previously reported [25-26]. In brief,

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all glasswares and PBS solution were purged with nitrogen gas prior to preparation. Then, 2

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M of sulfuric acid was added drop-wise to a saturated sodium nitrite solution, leading to the production of nitrogen oxide gas through disproportionation reaction of sodium nitrite in the

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acidic solution. The gas produced was bubbled through two KOH solutions of decreasing concentration (0.1 g/mL and 0.025 g/mL) in order to remove other forms of nitrogen oxides. Finally, NO gas was collected in PBS solution and stored in nitrogen-protected environment. The saturated NO solution at room temperature is reported to be 1.8 mM [25-26].

2.4. Fabrication of AuNPs-ERGO thin-film coated electrode

A polished glassy carbon electrode (GCE) was immersed consecutively in 100% ethanol and deionized water with sonication, followed by blow-drying with nitrogen gas. Graphite oxide was prepared using modified Hummer’s method [27], and sonicated for two 5

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hours to exfoliate graphene oxide (GO) sheets. GO solution is diluted to 0.25mg/mL and used for electrophoretic deposition (negatively charged GO sheets are electrostatically attracted onto the positively biased electrode). To electrophoretically deposit GO film on GCE, the

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electrode was immersed in the GO solution (0.25 mg/mL) with 2 V applied by an electrochemical station for 75 seconds. After the GO deposited electrode being dried in

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vacuum, GO film was electrochemically reduced to form ERGO film by cyclic voltammetry scanning in 1M KCl solution (0 to -1.2 V at 50 mV/s scan rate, for 20 cycles) [28]. Finally,

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AuNPs were in-situ synthesized onto ERGO film by applying -50 mV for 90s to the electrode

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immersed in a solution containing 1 mM HAuCl4 and 0.5 M sulfuric acid. For comparison, the same protocol was used to fabricate GCE electrode coated with only ERGO film or

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AuNPs.

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2.5 Experiments on HUVEC cells

Human umbilical vein endothelial cells (HUVECs, cell line obtained from Lonza)

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were cultured in MCDB131 medium supplemented with fetal bovine serum (10% v/v) and bovine brain extracts (0.2% v/v). After reaching confluence, the cells were harvested and suspended in PBS (6.60×105 cells per mL) for experiments. Using an AuNP-ERGO modified working electrode, the release from these cells (200 µL cell suspension) was amperometrically determined, without or with stimulation by acetylcholine.

3. Results and discussion

3.1. The AuNP-ERGO hybrid electrode

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Usually, graphene modified electrodes are made by drop-casting GO dispersion onto the supporting electrode followed by chemical reduction of GO [29-30]. Here, we deposit GO sheets onto a positively-biased glassy carbon electrode via electrophoresis because GO

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nanosheets bear abundant negatively charged hydroxyl, ketone carbonyls, epoxide, and carboxyl groups [31]. As compared to drop-casting and other coating methods,

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electrophoretic deposition can easily and reproducibly make uniform and continuous film with thickness controllable simply by timing [32-33]. Subsequently, the deposited GO is

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electrochemically reduced to ERGO by cyclic voltammogram scanning [29, 34]. In

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comparison with the usual chemical reduction of GO using harsh or hazardous chemicals (most notably, hydrazine), electrochemical reduction is environmental friendly and fast [34].

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As revealed by scanning electron microscopy (SEM) (Fig. 1A), the resulting ERGO film is a continuous network of micron-sized individual sheets with numerous wrinkles and

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edges which are believed to facilitate electron transfer [15]. Finally, gold nanoparticles

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(AuNPs) were decorated onto the ERGO film by in-situ electrochemical deposition, in contrast to the previously demonstrated spontaneous chemical reduction of AuNPs on RGO

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sheets [35]. As shown in Fig. 1B, AuNPs with an average size of ~50 nm and their small clusters uniformly disperse on the ERGO film. The identity of AuNPs is also confirmed by energy-dispersive X-ray spectroscopy (EDX) (Fig. 1C) which shows the atomic ratio of C:Au of ~ 3.1:1 and UV-vis absorption spectrum (Fig. 1D) which shows the absorption peak at 615 nm attributable to the surface plasmon effect of AuNPs [36]. The absorption peak of AuNPs is broadened and red-shifted due to the aggregation of AuNPs which leads to sharing of conduction electron between particles and hence lowering energy of the surface plasmon resonance [37].

Figure 1 7

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3.2. Comparison between the different electrodes by cyclic voltammetry

The cyclic voltammograms (CVs) of bare glassy carbon electrode (GCE) without or

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with high-voltage treatment (2V treated GCE) and GCEs modified with ERGO sheets, AuNPs or AuNP-ERGO hybrid film were measured in PBS in the absence or presence of 200

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M nitric oxide. As shown in Fig. 2 and summarized in Table 1, nitric oxide oxidation (via

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the following reactions: NO − e− → NO+; NO+ + OH− → HNO2; HNO2 → H+ + NO2− [3839]) can be observed from all the electrodes but AuNP-ERGO electrode shows largest

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oxidative current and lower oxidation potential than that of GCE, GO, or ERGO electrode. Specifically, as compared with the bare GCE, the AuNP-ERGO modified GCE electrode

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gives an increase of 53.9 μA in peak oxidative current and a reduction of 0.085 V in

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overpotential (averaged from three different electrodes, Table 1). It is arguable that GCE may be oxidized by the high voltage (2V for 75s) used to deposit GO thereby enhancing the

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electrode sensitivity [40]. This possibility is ruled out by the observation that the 2V-treated GCEs didn’t exhibit enhanced catalytic activity towards NO oxidation (Fig. 2) or morphological alteration (Fig. S1 in Supporting Information). The better performance of the AuNP-ERGO hybrid electrode suggests the synergistic integration of ERGO network and AuNPs: the former provides large active surface area for charge transfer and interconnected conducting pathways while the latter provides high catalytic activity towards NO [36].

Figure 2

Table 1 8

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3.3 NO detection

Figure 3

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CVs of the AuNP-ERGO electrode obtained at different NO concentrations (0 to 200 μM)

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show that the oxidation current scales linearly with the NO concentration (Fig. 3). In comparison to CV based measurement, amperometric measurement is able to offer sensitive

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and real-time detection. Fig. 4A shows the amperometric current response of AuNP-ERGO electrode (at holding voltage = 0.8 V vs Ag/AgCl) to successive addition of NO to reach

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various final concentrations. As shown, the amperometric response to 133 nM NO can be clearly resolved with a signal-to-noise-ratio (S/N) of ~5.5. The response time (the time taken

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to reach 95% of the steady state current) is ~3 s, which is shorter than the physiological

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lifetime (~ 5 s) of nitric oxide. In comparison, the amperometric response of GCE electrode

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decorated with only AuNPs is poor (Fig. 4A) suggesting the critical roles of ERGO sheets. This can be partly attributed to that AuNP deposition on ERGO coating is much denser than on bare GCE electrode (Fig. S2 in SI) because the oxygen-containing functional groups and defects on ERGO serve as the nucleation sites to facilitate formation of AuNPs [41-42].

Figure 4

The dose response curve (steady-state amperometric response vs. NO concentration) is plotted Fig. 4B. As seen, the detection range is wide with a lower experimental detection limit of ~133 nM, and the linear response is up to ~3.38 μM with a sensitivity of ~5.38 μA/μM/cm2. Such performance is better compared to the previously reported electrodes 9

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modified with RGO [29], AuNP array [21], AuNP-polyelectrolyte hybrid film [22], or AuNPCNT-polyelectrolyte composite [19]. However, the lower detection limit of our electrode is higher than that of a previously demonstrated electrode (0.31 nM) modified with fused

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spherical nanoparticles using simple sol-gel pr cess [20]. Furthermore, we show in Fig. 4C that NO detection by our hybrid electrode is insensitive to the common physiological

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interferences including oxalate, glucose, uric acid (UA), sodium ions, and ascorbic acid (AA). The selectivity against the negatively charged UA and AA molecules can be explained by the

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electrostatic repulsion by the negatively charged reduced graphene oxides and AuNPs.

materials,

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Electrodes with excellent performance on NO detection have been reported based on other alizarin-red

functionalized

carbon

nanofibers

[43],

functionalized

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microperoxidase functionalized carbon-nanotube nanocomposites [44] and hemoglobin nanomatrix

consisting

of

chitosan,

graphene

and

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hexadecyltrimethylammonium bromide [14]. These methods, however, require complicated

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fabrication procedures or expensive and fragile enzymes.

3.4 Detection of dynamic release of NO from HUVEC cells upon stimulation

The emerging nanoengineered electrodes or devices have brought new possibilities to

resolve dynamic cell functions [45-47]. Here, we demonstrate the use of our AuNP-ERGO hybrid electrode for real-time electrochemical detection of NO release from human umbilical vein endothelial cells (HUVECs) in response to acetylcholine stimulation. It is known that acetylcholine can acutely stimulate NO production and secretion from endothelial cells via activation of Ca2+-calmodulin dependent signaling cascade. [48] 10

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Using the experimental setup as shown in Fig. 5 A, injection of 2 mM acetylcholine into the HUVEC cell suspension in PBS (6.60×105 cells / mL) causes almost instantaneous increase of NO concentration (within 1 s) which is followed by a slower rise and subsequent

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decay after a few seconds (Fig. 5B). The peak current response (1.67 μA) corresponds to 3.99 μM increase of NO as the result of 2 mM acetylcholine stimulation is estimated based on the

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dose response curve shown in Fig. 4 B; subsequently, peak current response (201 nA) corresponds to 149 nM of NO as result of 0.5 mM acetylcholine stimulation. Apparently,

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acetylcholine stimulated NO release from HUVECs is an acute and potent process. In

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comparison, the response triggered by a lower dose of acetylcholine (0.5 M) is significantly reduced and the electrode is not responsive to the addition of PBS solution (Fig. 5B).

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Figure 5

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4. Conclusion

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In summary, we present a method to electrochemically prepare a hybrid thin-film

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electrode made of electrochemically reduced graphene oxide (ERGO) and gold nanoparticles (AuNPs). Comparing to the commonly used methods which usually involve functionalization of graphene oxides (GOs), drop-casting GOs onto the supporting electrode, and reduction of GOs using harsh chemicals [29-30, 49], the herein reported method is simple, fast, environmental friendly, and able to reproducibly fabricate uniform thin film. ERGO network provides highly conductive pathways for electron conduction and a

large surface area for catalyst support, while AuNPs act as efficient electrocatalysts towards the oxidation of nitric oxide. The synergistic integration of ERGO and AuNPs realizes the electrochemical detection of nitric oxide (NO) with high sensitivity (5.38 μA/μM/cm2), low experimental detection limit (133 nM with a S/N = ~5.5), and a fast response time (3 s). We 11

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further demonstrate that the AuNP-ERGO hybrid electrode can be used to electrochemically detect dynamic release of NO in response to acetylcholine stimulation from live human umbilical vein endothelial cells with high temporal resolution, suggesting its potential as tool

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to study the fundamental NO signaling processes.

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Acknowledgments

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This study is supported by AcRF Tier 2 grants from Ministry of Education of

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Singapore (MOE2011-T2-2-010, MOE2012-T2-2-049). We also thank GlobalFoundries

Appendix A. Supplementary data

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(Singapore) for the scholarship provided to S.L. Ting.

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References

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http://dx.doi.org/xxxxxx.

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Supplementary data associated with this article can be found, in the online version, at

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[41] G. Goncalves, P.A.A.P. Marques, C.M. Granadeiro, H.I.S. Nogueira, M.K. Singh, J. Grácio, Surface Modification of Graphene Nanosheets with Gold Nanoparticles: The Role of Oxygen Moieties at Graphene Surface on Gold Nucleation and Growth, Chemistry of Materials, 21 (2009) 4796-4802. [42] H.Y. Koo, H.-J. Lee, Y.-Y. Noh, E.-S. Lee, Y.-H. Kim, W.S. Choi, Gold nanoparticle-doped graphene nanosheets: sub-nanosized gold clusters nucleate and grow at the nitrogen-induced defects on graphene surfaces, Journal of Materials Chemistry, 22 (2012) 7130-7135. [43] D.Y. Zheng, X.J. Liu, H.M. Cao, S.Y. Zhu, Y.G. Chen, An Electrochemical Microsensor for the Detection of Nitric Oxide, Analytical Letters, 46 (2013) 790-802. [44] A.A. Abdelwahab, W.C.A. Koh, H.B. Noh, Y.B. Shim, A selective nitric oxide nanocomposite biosensor based on direct electron transfer of microperoxidase: Removal of interferences by co-immobilized enzymes, Biosensors & Bioelectronics, 26 (2010) 10801086. [45] Y.X. Huang, H.G. Sudibya, D.L. Fu, R.H. Xue, X.C. Dong, L.J. Li, P. Chen, Labelfree detection of ATP release from living astrocytes with high temporal resolution using carbon nanotube network, Biosensors & Bioelectronics, 24 (2009) 2716-2720. [46] Y.X. Huang, D. Cai, P. Chen, Micro- and Nanotechnologies for Study of Cell Secretion, Anal. Chem., 83 (2011) 4393-4406. [47] Y.X. Huang, P. Chen, Nanoelectronic Biosensing of Dynamic Cellular Activities Based on Nanostructured Materials, Advanced Materials, 22 (2010) 2818-2823. [48] R.A. Cohen, P.M. Vanhoutte, ENDOTHELIUM-DEPENDENT HYPERPOLARIZATION - BEYOND NITRIC-OXIDE AND CYCLIC-GMP, Circulation, 92 (1995) 3337-3349. [49] Y. Chen, Y. Li, D. Sun, D.B. Tian, J.R. Zhang, J.J. Zhu, Fabrication of gold nanoparticles on bilayer graphene for glucose electrochemical biosensing, Journal of Materials Chemistry, 21 (2011) 7604-7611.

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Table 1

Average Oxidation

Current Density

Current (µA)

Potential (V)

(mA/cm2)

AuNPs

128.3 ±11.5

0.796 ±0.001

AuNP-ERGO

143.5 ±11.7

0.821 ±0.018

2.021 ±0.164

ERGO

108.1 ±15.2

0.845 ±0.025

1.523 ±0.214

GO

61.8 ±0.6

1.08 ±0.056

0.871 ±0.008

0.970 ±0.007

1.227 ±0.046

0.906 ±0.011

1.262 ±0.096

cr

Average Peak

M

an

us

1.806 ±0.162

87.1 ±3.3

Ac ce p

2V treated GCE

te

d

Modifications

ip t

Summary of CVs from different electrodes from the experiments shown in Fig. 2.

GCE

89.6 ±6.8

*data is mean +/- standard deviation obtained from 3 independent experiments

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FIGURE CAPTIONS

ip t

Fig. 1. (A) SEM image of ERGO film. Inset shows an ERGO sheet. (B) SEM image of AuNP-ERGO film. Inset shows individual AuNPs with a high magnification. (C) EDX

cr

spectrum. (D) UV-vis absorbance spectra of AuNP-ERGO and ERGO film.

us

Fig. 2. The CVs of bare glassy carbon electrode (GCE) without or with high-voltage

an

treatment (2V treated GCE) and GCEs modified with ERGO, AuNPs or AuNP-ERGO hybrid

M

film, measured in PBS in the presence or absence (inset) of 200 µM nitric oxide.

Fig. 3. CV scans with increasing concentration of NO from 25 μM to 0.2 mM (arrow

d

indicates the direction of increasing concentration). Inset shows the peak CV current vs. NO

te

concentration fitted by a line with a slope of 0.921 μA/μM.

Ac ce p

Fig. 4. (A) Amperometric response of AuNP-ERGO electrode and GCE electrode decorated with only AuNPs (biased at 0.8 V vs Ag/AgCl electrode) to successive addition of NO to various concentrations. The response to 1.2 μM NO is displayed in the inset in an enlarged view. (B) Amperometric current responses vs. NO concentration of AuNPs-ERGO electrode and AuNPs coated electrode (averaged from 3 electrodes; the error bars indicate the standard deviations). The fitted lines in the linear response range have a slope of 0.382 and 0.095 μA/μM, respectively. (C) Amperometric responses to 10 μM NO, oxalate, glucose (Glu), uric acid (UA), sodium chloride, or ascorbic acid (AA).

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Fig. 5. (A) Photo and illustration of the experimental setup for cell experiments. (B) Amperometric response from HUVECs stimulated with acetylcholine of various concentrations. Bias voltage = 0.8 V. Arrow indicates the injection of stimulation solution.

Ac ce p

te

d

M

an

us

cr

ip t

Inset shows the cells grown in the culture flask.

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