Analytica Chimica Acta 804 (2013) 98–103

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A novel Au–Ag–Pt three-electrode microchip sensing platform for chromium(VI) determination Dongyue Li a,b , Jing Li a , Xiaofang Jia a,b , Yong Xia a,∗ , Xiaowei Zhang a,b , Erkang Wang a,b,∗ a State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China

h i g h l i g h t s

g r a p h i c a l

• A rapid and portable electrochemical

The as-prepared lab-on-a-chip integrated electrochemical detector performed with high sensitivity and good reproducibility in Cr(VI) determination. Combined with custom USB electrochemical system (␮ECS), this sensor presents advantages of portability, ease of use, low analyte consumption, low cost, fast response time and demonstrates great potential for the application of high-throughput and in-field environmental monitoring Cr(VI) pollutant.

microchip sensor for Cr(VI) has been constructed. • Au–Ag–Pt three-electrode system was integrated on PMMA substrate. • The as-prepared detector performed with high sensitivity and good reproducibility.

a r t i c l e

i n f o

Article history: Received 24 June 2013 Received in revised form 8 October 2013 Accepted 8 October 2013 Available online 17 October 2013 Keywords: Microchip sensor Gold–silver–platinum three-electrode system Electrochemical detection Chromium(VI)

a b s t r a c t

a b s t r a c t A simple, rapid and portable electrochemical microchip sensing platform has been successfully constructed for chromium(VI) determination. Gold–silver–platinum (Au–Ag–Pt) three-material electrodes (gold as working electrode, silver as reference electrode and platinum as counter electrode) were integrated on one poly(methyl methacrylate) (PMMA) substrate by polymer compatible photolithography process. The three-electrode microchip sensing platform was used for Cr(VI) determination for the first time, and exhibited high sensitivity and good reproducibility. A wide linear range from 2 to 200 ␮M with a good linear correlation (R2 = 0.998) was obtained, and the detection limit was 0.9 ␮M. In addition, the practical analytical application of the sensing micro-platform was assessed by determination of Cr(VI) in real water samples with satisfactory results. Armed with the remarkable advantages, such as ease of use, low analyte consumption, inexpensive cost and fast response time, the microchip sensing platform may hold great potential for the high-throughput and in-field environmental monitoring Cr(VI) pollutant. © 2013 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding authors at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China. Tel.: +86 431 85262003; fax: +86 431 85689711. E-mail addresses: [email protected] (Y. Xia), [email protected] (E. Wang). 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.10.014

Chromium(VI) is a widespread pollutant in the environment due to the disposal of industrial wastes from chrome-plating industries, dyes/pigments manufacture and leather tanning. Cr(VI) is believed to pose severe adverse effects on human health through water consumption because of the potential carcinogenic effect on human health [1,2]. For example, ingestion of a considerable amount of

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Cr(VI) can lead to stomach ulcers, pulmonary congestions, convulsions, liver and kidney damage and dermatitis [3,4]. Accordingly, exploring the sensitive, rapid and portable sensing platform for precise monitoring of Cr(VI) is urgently needed. To date, a range of analytical techniques for Cr(VI) determination has been developed including atomic absorption spectrometry (AAS) [4,5], inductively coupled plasma-mass spectrometry (ICP-MS) [6], UV–vis spectrophotometry [7], atomic fluorescence spectrometry (AFS) [8,9] and electrochemical techniques (EC) [10–18]. Among all the methods, EC techniques stand out as a powerful tool for the determination of Cr(VI) due to their inherent sensitivity and selectivity coupled with the ease of the miniaturization and portability for real-time analysis. The electrochemical detection of Cr(VI) has been successfully reported in connection with various solid electrodes, such as glassy carbon [10], carbon paste [12], gold, platinum [14] as well as different chemical and physical modifications of these electrodes [19–22]. Adsorptive striping voltammetry was often carried out to measure Cr(VI) on carbon electrodes [17,22], however, additional equipment, such as a rotator or a stirrer, was required and led to difficulty in the integration on the chip. Moreover, most of these methods for Cr(VI) detection were performed only in the laboratory and few of them were used for in-field analysis. To develop the integrated and portable electroanalytical sensing platform, the planar electrodes were generally constructed on a substrate using microfabrication and screen-printing techniques. Combing integrated electrodes with the microfluidic components as a lab-on-chip device provided a portable and miniaturized platform to achieve in-field assays of heavy metal ions. For example, Ahn’s research group developed micro electrochemical biosensors with integrated on-chip electrodes and microfluidic channels for monitoring of Pb(II) and Cd(II) [23,24]. Our group developed a new two-step photolithography fabrication method and built up the two-metal electrode system on a glass slide for Hg(II) sensing [25,26]. Nie et al. [27] investigated the microfluidic paper-based electrochemical sensing chips for selective analysis of Pb(II) in environmental aqueous solution. Chen et al. [26] made an attempt of using DNA-modified gold chip electrode for voltammetric detection of Hg(II). However, the complex modification procedures were inevitable unavoidable and limited their further practical application. Recently, Liu et al. [28] developed a new method for the fabrication of a monolithic, integrated Au–Ag–Pt electrode on poly(methyl methacrylate) substrate using a polymer compatible photolithography process. As an alternative to the traditional glass substrates, polymer provided a good opportunity for the miniaturized platform due to the favorable properties of biocompatibility, inexpensiveness and not as fragile as glass [23]. In previous work, Compton and co-workers [11] demonstrated that the direct reduction of Cr(VI) preferred to occur on a Au electrode compared to carbon electrode. Inspired by these works, a simple, rapid and portable electrochemical microchip sensor for Cr(VI) has been established with Au–Ag–Pt three-electrode system. For the first time, the as-prepared lab-onchip integrated electrochemical detector was performed for Cr(VI) determination with high sensitivity and good reproducibility. One group of microelectrodes and one chamber compose one sensing unit. Each group of microelectrodes consists of a gold working electrode, an Ag reference electrode and a platinum counter electrode. All the microchip sensor units on one PMMA substrates were single use and the reproducibility of three random microchip sensing units for Cr(VI) trace analysis was 4.8%. Combined with custom USB electrochemical system (␮ECS), this sensor presented advantages of portability, ease of use, low analyte consumption, low cost, fast response time and demonstrates great potential for the application of high-throughput and in-field analysis of environmental Cr(VI).

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2. Experimental 2.1. Reagents All chemicals were analytical grade, or better, and used as received. Unless otherwise stated, all solutions were prepared with pure water from Water Purifier (Sichuan Water Purifier Co., Ltd., China). BP212 positive photoresist was purchased from Beijing Institute of Chemical Reagents, China. Cr(VI) were prepared by dissolving K2 Cr2 O7 in twice-distilled water. Prior to use, stock solutions were diluted to the desired concentration with a supporting electrolyte. 1,5-Diphenylcarbazide (DPC) was purchased from Sinopharm Chemical Reagent Co., Ltd. DPC as a selective reagent is classically utilized in the qualitative and quantitative analysis of Cr(VI). In this work, 0.1 M HCl was employed as the supporting electrolyte. All the solutions were used under the condition of open air without removing the dissolved oxygen. 2.2. Apparatus Cyclic voltammetry (CV) and differential pulse cathodic stripping voltammetry (DPCSV) were carried out with a custom USB 2.0 electrochemical system (␮ECS) (Scheme 1B). The ␮ECS is the newly invented electrochemistry researching system that can be connected with computer by USB, which also allows the convenience for educational presentation and hi-tech scientific researching. The ␮ECS is home-made in our laboratory and now can be commercially available from State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry by contacting Dr. Yong Xia. The electrochemical microchip sensor consists of a gold working electrode (2.0 mm diameter), an Ag reference electrode and a Pt counter electrode on a PMMA substrate (Scheme 1C). Dimensions of both the counter electrode and the reference electrode are 1 mm × 1.5 mm. A conventional three-electrode system was employed, consisting of an Au solid electrode (2.0 mm diameter), a Ag/AgCl (saturation KCl) electrode as the reference electrode and a platinum wire as the auxiliary electrode. The Au solid electrode was polished with aqueous slurries of successively finer alumina powder (1.0 ␮m and 0.3 ␮m), sonicated for 3 min in ethanol and pure water, respectively. All electrochemical experiments were carried out at room temperature. UV–vis absorption spectra was recorded by a CARY 500 UV-vis-near-IR Varian spectrophotometer. 2.3. Fabrication of Au–Ag–Pt three-electrode system Fabrication of Au–Ag–Pt three-electrode referred to our previously published work [28]. Briefly, Ag reference electrode layer (200 nm) was first vaporized on the PMMA substrate with Ti as the adherent layer (20 nm). The patterned Ag reference electrode was obtained under the cover of photomask and exposed Ag which was removed with NH4 OH and H2 O2 (5:2, v/v). Titanium adherent layer was then removed by hydrofluoric acid, while BP212 positive photoresist was removed with 0.5% NaOH solution to avoid the dissolution of PMMA. The gold working electrode and the platinum counter electrode were then fabricated using a lift-off process and the thickness of both was 100 nm [29]. The microchannel layer of the microfluidic device is fabricated in poly(dimethylsiloxane) (PDMS) using a normal phototyping technique [25]. 2.4. Analytical procedure The Au electrode was electrochemically pretreated in 0.1 M H2 SO4 solution by repeating the potential scan from −0.35 to +1.4 V at 0.4 V s−1 (vs. Ag reference on-chip) until CV characteristics for a clean gold electrode were obtained. The DPCSV of Cr(VI) was recorded by applying a negative-going potential scan from 0.9

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Scheme 1. Schematic representation of the microchip sensor system (A) containing custom USB electrochemical system (B) and Au–Ag–Pt microchip on PMMA substrate (C).

to −0.2 V (with a step increment of 0.005 V, amplitude of 0.08 V, pulse period of 0.05 s) in 0.1 M HCl. A chrome-plating industry sewage sample was collected from Guangzhou (China). Tap-water was collected from the laboratory. The domestic sewage sample was supplied from a municipal wastewater treatment and a small proportion of industrial wastewater from an automobile factory plant in Changchun. Avoiding interference of large size particles, all the sewage samples (liquid) were filtered through a 0.22 ␮m membrane (Millipore) by a 5 mL syringe. Then the samples were diluted with 1 M HCl before voltammetric measurements. Last, a PDMS chamber was used to cover the as-prepared electrochemical chip, 50 ␮L samples were added into chamber and voltammogram was recorded by applying a negative-going potential scan from 0.9 to −0.2 V.

3. Results and discussion

Fig. 1. Cyclic voltammogram of 50 cycles with gold working electrode on the chip in 0.1 M H2 SO4 . Potential range: −0.35 V to +1.4 V (vs. Ag reference on-chip), scan rate: 0.4 V s−1 .

3.1. Characterization of the microchip Au–Ag–Pt system The electrochemical sensing microchip consists of a gold working electrode, an Ag reference electrode and a Pt counter electrode on a PMMA substrate. Electrochemical experiments were carried out to characterize the Au–Ag–Pt three-electrode system. Cyclic voltammetry was applied with a potential between −0.35 V and +1.4 V (vs. Ag reference on-chip) to the gold electrode using 0.1 M H2 SO4 as the supporting electrolyte, conducted with a high scan rate of 0.4 V s−1 . With one sensing unit, the voltammograms of 50 cycles were given in Fig. 1. Besides the sharp peak of the reduction of gold oxide at +0.6 V, a series of multiple overlapping peaks of gold oxidation was also found in the range of +0.9 to +1.2 V, which is consistent with the results obtained from Au–Ag–Au three-electrode systems fabricated on glass substrates [26] and indicates the three-electrode system fabricated here can perform well in electrochemical detection. Reproducibility was an essential characteristic of the fabricated three-electrode system. With one microchip sensing unit, the relative standard deviations (RSDs) of peak current and peak potential for the reduction of gold oxide of 50 cycles were 0.11% and 0.74%, respectively (Fig. 1). Furthermore, the

reproducibility of three random sensing units was characterized and the RSDs of peak current and peak potential for the reduction of gold oxide were 1.74% and 0.32%, respectively (Table 1). The excellent reproducibility of the three-electrode system integrated on PMMA substrates is critical to the in-field environmental application.

Table 1 Reproducibility of three random sensing units from the microchip. Unit

1 2 3 RSD (N = 3)

Reduction of gold oxide

Reduction of Cr(VI)

Potential (V)

Current (␮A)

Potential (V)

Current (␮A)

0.601 0.597 0.600 0.32%

104.6 108.1 105.2 1.74%

0.328 0.315 0.320 2.1%

8.757 7.994 8.191 4.8%

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Fig. 2. (A) DPCSV responses of 50 ␮M Cr(VI) on a Au solid electrode (vs. Ag/AgCl reference) in 0.1 M HCl (solid line), 0.01 M HCl (dashed line) and 0.001 M HCl (dotted line). (B) DPCSV responses of 100 ␮M Cr(VI) vs. Ag reference on-chip (solid line) and 60 ␮M Cr(VI) vs. Ag/AgCl reference of Au solid electrode (dashed line) in 0.1 M HCl. DPV scan with a step potential of 0.005 V; amplitude of 0.08 V; pulse period of 0.05 s.

3.2. Effect of the varying concentrations of hydrochloric acid solution The electrochemical reduction of hexavalent chromium on Au electrode has been reported to be proton-dependent process [11] and the effect of different concentrations of HCl solution toward to Cr(VI) sensing is shown in Fig. 2A and indicates the DPCSV responses of 50 ␮M Cr(VI) on a Au solid electrode (vs. Ag/AgCl reference) in 0.1 M HCl (solid line), 0.01 M HCl (dashed line) and 0.001 M HCl (dotted line), respectively. It can be seen that the electrode response in high concentration of HCl (0.1 M) produced a well-defined reduction peak at 0.45 V (Fig. 2A solid line) when compared to more diluted concentrations of HCl. And the peak potential of Cr(VI) shifts into more positively with an increase of the acid concentration as reported in the previous work [30]. In 0.1 M HCl, the primary species is HCrO4 − and the electrochemical reduction of Cr(VI) is via a one-electron and one-proton reaction followed by disproportionation (described as follows). In 0.001 M HCl, Cr(VI) exhibited a very weak reduction signal at 0.25 V due to the low concentration of H+ . With the increase of HCl greater than 0.1 M (not shown), a high background signal was obtained. Therefore, 0.1 M HCl was used due to high sensitivity and low background noise. HCrO4 − + e− + H+  H2 CrO4 −

2Cr(V)  Cr(IV) + Cr(VI)

(slow)

3.3. Reproducibility of the microchip Reproducibility is one of the most important performances to evaluate the capability of the microchip sensor. With one sensing unit, a series of 10 repeated determinations of two different concentrations of Cr(VI) were used for demonstrating the reproducibility of microchip electrode in Fig. 3, respectively. The voltammogram was recorded by applying a negative-going potential scan from 0.9 to −0.2 V in 0.1 M HCl. The obtained data are calculated with RSDs of 3.9% and 6.3%, respectively, for 20 ␮M and 100 ␮M Cr(VI), which are also comparable to those obtained from the three-electrode system on glass substrates [25]. Furthermore, the reproducibility of three random sensing units from the microchip was summarized in Table 1. The peak currents and peak potential of measuring 100 ␮M Cr(VI) in the HCl with 3 different sensing units were with RSDs of 4.8% and 2.1%, respectively. In conclusion, excellent reproducibility was achieved by such a three-metal electrode system. 3.4. DPCSV for Cr(VI) analysis on microchip A PDMS frame chamber was used to cover the as-prepared electrochemical chip, then 50 ␮L samples with various concentrations of Cr(VI) were added into chamber. The voltammogram was recorded by applying a negative-going potential scan from 0.9 to −0.2 V in 0.1 M HCl. Fig. 4 shows the DPV scan curves at different concentrations of Cr(VI). The peak current of each concentration

(fast)

Cr(IV) + Cr(V)  Cr(III) + Cr(VI)

(fast)

or 2Cr(IV)  Cr(III) + Cr(V)

(fast)

Under the optimal conditions, the analytical performance of the prepared chip for Cr(VI) was investigated. As depicted in Fig. 2B, the reduction peak potential of Cr(VI) was found at ca. 0.320 V vs. Ag reference electrode on chip (solid line). The reduction peak potential of Cr(VI) was found at ca. 0.439 V vs. Ag/AgCl reference electrode on Au solid electrode (dashed line). The 0.119 V peak shift observed was attributed to the different potential of reference electrodes adopted. As has been previously shown, the difference between Ag and Ag/AgCl electrode potential is 0.114 V [25]. Thus, the potential obtained at 0.320 V vs. Ag reference electrode demonstrated the integrated three-electrode system electrode performed well with good accuracy.

Fig. 3. Reproducibility for peak current responses of 20 ␮M and 100 ␮M Cr(VI) samples. The signals were detected 10 times at different times under the conditions as mentioned previously.

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Fig. 4. (A) Stripping voltammograms for the different concentrations of Cr(VI) (2, 4, 10, 15, 40, 50, 60, 100, 150, 200 ␮M) with gold working electrode on-chip in 0.1 M HCl. (B) The corresponding calibration plot. The error bars represent standard deviations of three successive measurements.

was obtained from an average of three detection readings. We obtained linear responses of the peak current against the concentrations of Cr(VI) over the ranges of 2–200 ␮M. The calibration curve was y = 0.3887 + 0.0753 x, R2 = 0.998 (x: concentration/␮M, y: current/␮A). Based on three times of the background noise, the detection limit was 0.9 ␮M 47 ppb, which reached the guideline value set by World Health Organization (WHO) for Cr(VI) in groundwater (50 ppb).

Table 2 Recovery of the Cr(VI) added in different practical samples. Samples

Added (␮M)

Recovery (%)

RSD (%) (N = 3)

Tap water

10 20 40 60 40 50

102.15 96.99 98.92 97.44 98.61 96.42

1.78 2.54 1.01 0.45 1.11 2.50

Domestic sewage Industrial sewage

3.5. Application To further demonstrate the practicality of the microchip electrode, chrome-plating industry sewage water, tap water and sewage samples were collected and tested. Fig. 5 shows the DPCSVs for the chrome-plating industry sewage sample and each addition of 20 ␮M Cr(VI) to the sample, respectively. A well defined peak around 0.38 V was observed and increased proportionally with the addition of Cr(VI). The concentration of Cr(VI) in the chrome-plating industry sewage water was 2.13 ␮M using the standard addition method. We employed diphenylcarbazide spectrophotometric method (GB 7467-87) to evaluate the concentration of the Cr(VI) in the chrome-plating water. Diphenylcarbazide as a selective reagent is classically utilized in the qualitative and quantitative analysis of Cr(VI) and the DPC colorimetric sensor for Cr(VI) as a standard method is widely used. The concentration of Cr(VI) obtained with DPC colorimetric method was 1.92 ␮M (present in Fig. S1) which was consistent with the data using the electrochemical method. For the sample of tap water and sewage

samples, no Cr(VI) was detected. Therefore, a recovery test was carried out by spiking a known concentration of Cr(VI) (shown in Table 2) into the water samples in order to verify the feasibility of this entire method. Satisfactory results were achieved in tap water and sewage water (present in Table 2). The recoveries in different water samples were from 96.42 to 102.15% with the RSDs of 0.45–2.54%, indicating the great potential applicability of the microchip sensors for Cr(VI) quantification in real samples. 4. Conclusions A novel Au–Ag–Pt three-electrode microchip sensor has been successfully constructed for chromium(VI) determination on a PMMA substrate. The three-electrode system exhibited excellent reproducibility, and the hybrid PMMA/PDMS electrochemical sensing microchip showed great potential for in-field trace level Cr(VI) ion analysis. The practical application of the microchip sensor was verified in real water samples with satisfactory results. Combined with custom USB electrochemical system makes it suitable for in-field sample analysis due to the small size and fast response time. This kind of microchip sensor demonstrates great potential applications of in-field environmental monitoring chromium(VI) pollutant. Acknowledgements The authors would like to thank Junshan Liu for his kindly assistance with the chip. This work is supported by National Natural Science Foundation of China 21105094, 973 Project 2010CB933600, 863 Project 2013AA065601 and Jilin Province Science and Technology Development Plan Project 20130522130JH. References

Fig. 5. DPCSV signals for the determination of Cr(VI) in chrome-plating industry water sample spiked with 0, 20, 40, 60, 80, 100 ␮M on-chip, respectively.

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