Electrochemical determination of glutathione based on an electrodeposited nickel oxide nanoparticles-modified glassy carbon electrode

1

Electrochemical Determination of Glutathione Based on

Electrodeposited Nickel Oxide Nanoparticles Modified Glassy

Carbon Electrode

Baiqing Yuan a,

*, Xiaoying Zeng a, b

, Dehua Deng a, Chunying Xu

a, Lin Liu

a, Jiayu

Zhang a, Yan Gao

a, Huan Pang

a, c, *

a College of Chemistry and Chemical Engineering, Anyang Normal University,

Anyang 455000, Henan, China

b School of Pharmaceutical Science, Zhengzhou University, Zhengzhou 450001,

Henan, China

c State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing,

Jiangsu 210093, China

* Corresponding authors. Tel.: +86 0372 2900040.

E-mail: [email protected] (Baiqing Yuan); [email protected]

(Huan Pang)

Page 1 of 20 Analytical Methods

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View Article OnlineView Journal

http://dx.doi.org/10.1039/c3ay26536f

http://pubs.rsc.org/en/journals/journal/AY

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Abstract

The electrodeposited nickel oxide nanoparticles (NiONPs) modified glassy carbon

(GC) electrode was first presented for the electrochemical determination of

glutathione (GSH). The NiONPs/GC electrode showed low oxidation potential toward

GSH and wide linear range for the its analysis. The different forms of nickel oxide,

electrochemically synsisized in different pH buffer solution, were investigated for the

electrochemical oxidation of GSH. The presented sensor was also applied in the

analysis of GSH in the prescence of uric acid (UA). In addition, the effects of other

interfering species including ascorbic acid (AA), dopamine (DA) and glucose were

examined and disscussed in detail.

Keywords: Nickel oxide; Electrodeposition; Glutathione; Uric acid

1. Introduction

Reduced L-glutathione (GSH), an important thiol compound presented in most

mammalian cells, plays an essential role in many biological systems such as

catabolism and transportation [1, 2]. In addition, as a physiological antioxidant, GSH

can reduce oxidative stress in cells and maintain redox homeostasis that is crucial for

cell growth [3]. The concentration of GSH in living cells is at the level of several

millimol per liter [4], and the level of GSH in plasma has been directly linked to some

diseases, including Alzheimer’s, Parkinson’s diseases, diabetes, macular degeneration,

and HIV disease [5].

Numerous methods for the determination of GSH have been reported including

liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis

Page 2 of 20Analytical Methods

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(CE) coupled with various detectors, such as ultraviolet–visible spectroscopy (UV)

[6], fluorescence [7], mass spectrometry [8] and electrochemical detector [9, 10].

However, these separation methods suffer from difficulties or drawbacks in terms of

equipment cost, the need for derivatization, and sample preparation [3].

Electrochemiluminescence [11] and electrochemical detection [5, 10, 12-26] were

also demonstrated for its analysis. By contrast, electrochemical detection has the

advantages of simplicity, high sensitivity, high selectivity, and low instrumental cost.

The electrochemical determination of thiols was mainly based on some organic or

inorganic molecules and materials. These molecules included cobalt phthalocyanine

[27], piazselenole [3], pyrroloquinoline quinine [28], fluorone black [29], and

4,4’-biphenol [30]. The electrode materials employed for the determination of thiols

mainly relied on mercury [12], TiO2 nanoparticles [5], copper hydroxide [13],

CuGeO3 [14], Au nanoclusters [15], Ce-doped Mg-Al layered double hydroxide [16],

poly-m-aminophenol [17], PtFeNi [18], PtNiCo [19], and carbon based electrode

including edge plane pyrolytic graphite [20], boron doped diamond electrode [21],

fullerenes [22], carbon nanotubes [23, 24] and ordered mesoporous carbon [25, 26].

Among these materials, inexpensive and easily available materials are promising for

the electrochemcial detection of GSH. In addition, stable modification methods are

essential for good reproducibility.

The inexpensive nickel oxide nanomaterial casted glassy carbon electrode was first

presented for the electrochemical determination of GSH by Pumera’s group [4].

Recently, we synthesized the porous nickel oxide microflowers by calcination and


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prepared nickel oxide based carbon paste electrode for the electrochemical detection

of GSH with high sensitivity [31]. However, to the best of our knowledge,

electrochemical determination of GSH using electrodeposited nickel oxide has not

been reported. Herein nickel oxide nanoparticles (NiONPs) modified glassy carbon

(GC) electrode was fabricated by a modified electrochemcial method. The presented

electrode exhibited low electrochemcial oxidation potential toward GSH, and the

electrodeposited NiONPs was stable on GC electrode.

2. Experimental

2.1. Chemicals and solutions

GSH, uric acid (UA), ascorbic Acid (AA), dopamine (DA), and nickel (II) nitrate

hexahydrate were purchased from Aldrich (Milwaukee, WI, USA). All other

chemicals used were of analytical reagent grade, and the aqueous solutions were

prepared with doubly distilled water. 0.1 M acetate buffer was used as the supporting

electrolyte for the electrochemical experiments.

2.2. Apparatus

Electrochemical measurements were performed with a CH Instrument model 842C

voltammetric analyzer (Austin, TX, USA) using a 3 mm GC electrode as working

electrode, a platinum coil as auxiliary electrode, and a Ag/AgCl electrode as reference

electrode, respectively. The Electrochemical impedance spectroscopy (EIS) was

measured in 5 mM [Fe(CN)6]3-/4-

with CH Instrument model 660D voltammetric

analyzer (Austin, TX, USA). The morphology of electrodes was directly examined by

field emission scanning electron microscope (SEM) (JSM-6701F, Jeol, Japan) using


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special GC electrode (3 mm) for SEM.

2.3. Preparation of NiONPs /GC electrode

Prior to modification, the bare GC electrode was polished to a mirror-like surface

with 1, 0.3, and 0.05 µm alumina slurry, respectively. The NiONPs/GC electrode was

structured by a modified electrochemical method including 3 steps [32]. First, the GC

electrode was scanned for 30 segments in acetate buffer (pH 4) using CV between

-0.5~1V. After that, the electrode was treated for 300 s at -0.85 V in the presence of 10

mM Ni2+

(pH 4). Finally, the treated electrode was scanned again as same as in step 1.

2.4. Sample analysis

The reduced GSH eye drops, purchased from Wuhan Wujing Medicine Co., Ltd,

was directly injected to the detection solution with microsyringe without pretreatment

and analyzed under stirring by amperometric i-t curve.

3. Results and discussion

3.1 Electrochemical, SEM, and EIS characterization of modified electrode

Electrodeposition technique has attracted a lot of attention in synthesizing various

nanomaterials especially in the field of nanomaterials based electrochemical sensors

because of its simplicity and easy control of the shape and thickness. NiONPs were

electrodeposited on the surface of GC electrode by a modified electrochemical

method [32] including first treating, electrodeposition of metallic nickel, and

transformation to nickel oxide. Figure 1 shows the first (curve a) and tenth (curve b)

CV curves of electrodeposited nickel on GC electrode in acetate buffer (pH 4). It can

be seen that two oxidative peaks are observed on the first CV curve (curve a) but not


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on the other subsequent CV curves, indicating that stable nickel oxide was formed on

the surface of electrode [33].

The morphology of bare GC (A) and NiONPs/GC (B) electrode was examined by

SEM (Figure 2). As can be seen from Figure 2, the electrodeposited NiONPs were

uniform, and the diameter of particles was about 40 nm. When more negative

potential was applied in step two, the NiONPs aggregated (not shown).

The Nyquist diagrams of bare GC and NiONPs/GC electrode were presented in

Figure 3. The Nyquist diagram of bare GC electrode showed the characteristic

semicircle and Warburg impedance. By contrast, when NiONPs were modified on the

surface of GC electrode, the semicircle diameter related to the electrochemical

process decreased, suggesting the electrodeposited NiONPs accelerates electron

transfer rate of [Fe(CN)6]3−

/4−

.

3.2 electrochemical oxidation of GSH

Figure 4 shows the CVs of NiONPs/GC electrode in the presence (solid line) and

absence (dotted line) of 5 mM GSH in 0.1 M acetate buffer (pH 5). The inset is the

CV of GC electrode in the presence of 5 mM GSH. It can be seen that the

electro-oxidation of GSH proceeds very slowly and no oxidation peak is observed at a

bare GC electrode. However, one obvious oxidation peak is observed at GC/NiONPs

electrode in the presence of GSH, and there is no oxidation peak at GC/NiONPs

electrode in the background electrolyte, indicating that the electrodeposited NiONPs

decreases the oxidation overpotential of GSH. The oxidation peak potential was at

about 0.36 V, which was lower than that obtained at Pd-IrO2 [10],


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poly-m-aminophenol [17], and nickel oxide [4] modified electrode, but higher than

that obtained at ordered mesoporous carbon/cobalt oxide nanocomposite modified

electrode [25] and nanoscale copper hydroxide composite carbon ionic liquid

electrode [13].The chemical formula of electrodeposited NiONPs is dependent on the

pH of the supporting electrolyte. The NiONPs electrodeposited in pH 4 acetate buffer

is a mixed nickel oxide (NiO(Ni2O3)) [33]. The NiONPs for the electro-oxidation of

GSH electrodeposited in different pH acetate buffer (pH 4～8) were investigated, and

the results showed that all of NiONPs could electrocatalyze the oxidation of GSH (not

shown). The catalytic process was supposed as follows.

Ni (Ⅱ) ⇄ Ni (Ⅲ) + e

Ni (Ⅲ) + GSH ⇄ 1/2GSSG + H+ + Ni (Ⅱ)

The effect of pH (pH 4～6) on the voltammetric responses of GSH at NiONPs/GC

electrode is presented in Figure 5. We can see that the voltammetric signal of GSH at

pH 5 is the highest, and the oxidation peak of GSH disappeared while the pH is

beyond 7. Therefore, pH 5 acetate buffer was selected as the background electrolyte

for the electrochemical determination of GSH.

3.3 Amperometric detection of GSH

Figure 6 shows the amperometric sensing of GSH by successive addition of 0.125

mM GSH at 0.35 V in pH 5 acetate buffer. The NiONPs/GC electrode demonstrated

fast response for the electro-oxidation of GSH. The RSD for 11 successive detection

of 0.125 mM GSH was 4.7%. The linear range for GSH is from 12.5 µM to 2.3 mM

(R2=0.9925, n=32) with the detection limit of 2 µM (Inset A), which is wider than that


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obtained at Pd-IrO2 (10~800 µM) [10], poly-m-aminophenol (0.1~5.0 µM) [17], and

NiO (0.2~6.0 mM) [4] modified electrode, and is comparable to that obtained at

porous NiO-CPE (0.01~6 mM) [27], but is inferior to that obtained at nanoscale

copper hydroxide composite carbon ionic liquid electrode (1 µM~1.8 mM) [13].

3.4 Interferences

In order to evaluate the anti-interference ability, the effects of common interfering

species, including ascorbic acid (AA), uric acid (UA), dopamine (DA) and glucose

were investigated were selected. Glucose cannot be electrochemically oxidized under

the acidic condition at the NiONPs modified GC electrode, and did not interfere on

the electrochemical detection of GSH. The oxidation potential of UA was much

higher than that of GSH, and the DPV responses of GSH with increasing

concentrations in the presence of 1 mM uric acid at NiONPs/GC electrode was

investigated (Figure 7). As can be seen from Figure 7, the electro-oxidation of uric

acid occurred at 0.34 V, and the oxidization peak appeared at 0.44 V. The oxidation

peaks of GSH and uric acid can be well separated, and the simultaneous determination

of the two substances can be achieved. The sensor exhibited a linear response range

from 50 µM to 4.56 mM with a correlation coefficient, R2=0.9925 (n=9) in the

presence of 1 mM uric acid.

In addition, the oxidation of AA, DA and GSH overlapped each other (not shown),

however, 5 µM AA and 5 µM DA did not interfere significantly on the determination

of 0.5 mM GSH. It was reported that the concentration of GSH in the cells can be up

to 10 mM [34]. Therefore, when this method was employed to determine the GSH in


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blood sample, the sample can be diluted in order to reduce the interference of AA and

DA.

3.5 Reproducibility and stability

The stability and reproducibility of NiONPs/GC modified electrode was also

examined. The steady-state response current of the NiONPs/GC modified electrode

retained 86% of its initial current response to GSH after 3000 s continuous

measurement with magnetic stirring. In addition, the long-term stability of the sensor

to GSH was also evaluated, and the amperometric current showed a loss of 5% after

the NiONPs/GC modified electrode was stored at ambient temperature for 30 days.

3.6 Application

The NiONPs modified electrode could be applied to assay the reduced GSH eye

drops (purchased from Wuhan Wujing Medicine Co., Ltd), and the average

concentration by five replicate measurements was 61 mM (RSD=4.6%, n=5), which

was in good agreement with the labeled value (65 mM).

4. Conclusions

NiONPs were electrochemically synthesized on the surface of GC electrode by

three steps including first treating, electrodeposition of metallic nickel, and

transformation to nickel oxide. The NiONPs/GC modified electrode showed excellent

electrocatalytic oxidation toward GSH and wide linear range for its determination.

The sensor was also used for the analysis of GSH in the presence of uric acid, and

good results were obtained.

Acknowledgments


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Financial supports from the National Science Foundation of China (Nos. 21205003

and 21201010) and Key Project of Science and Technology Department of Henan

Province (No. 122102310521) are gratefully acknowledged.

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Figure captions

Figure 1. The first (a) and the tenth (b) cyclic voltammetric responses of GC/Ni

electrode in acetate buffer (pH 4) at 0.1 V/s. Initial scan potential: -0.5 V.

Figure 2. SEM images of bare GC (A) and NiONPs/GC (B) electrode.

Figure 3. Nyquist diagram for NiONPs/GC electrode in 5 mM [Fe(CN)6]3−

/4−

(1:1)

solution containing 0.1 M KCl. Inset: Nyquist diagram for GC. The frequency range

of EIS was from 0.1 to 100 Hz at 0.25 V.

Figure 4. Cyclic voltammograms of NiONPs/GC electrode in the presence (solid line)

and absence (dotted line) of 5 mM GSH in 0.1 M acetate buffer (pH 5). Inset, cyclic

voltammogram of GC electrode in the presence of 5 mM GSH. Scan rate: 0.1 V/s.

Figure 5. Cyclic voltammograms of NiONPs/GC electrode in the presence of 5 mM

GSH in different pH acetate buffer. Dashed line: pH 4; solid line: pH 5; dotted line:

pH 6. Scan rate: 0.1 V/s.

Figure 6. Amperometric sensing of GSH by successive addition of 0.125 mM GSH at

0.35 V in pH 5 acetate buffer. Inset: (A), amperometric response with the

concentration of GSH from 12.5 µM to 2.3 mM. (B), amperometric response at low

concentration of GSH.

Figure 7. DPVs of GSH in the presence of 1 mM uric acid. GSH concentration from

bottom to top is 0, 0.05, 0.09, 0.22, 0.42, 0.54, 0.72, 1.2, 2.2, and 4.56 mM. Inset: (A),

amplification of the graph; (B), amperometric response with the concentration of GSH

from 50 µM to 4.56 mM.


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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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