Methylene Blue-Mediated Electrocatalytic Detection of Hexavalent

Feb 11, 2015 - We report, for the first time, the design and fabrication of an electrochemical ion (E-ION) sensor for highly specific detection of hex...
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Methylene Blue-Mediated Electrocatalytic Detection of Hexavalent Chromium Lee E. Korshoj, Anita J. Zaitouna, and Rebecca Y. Lai* Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, United States S Supporting Information *

ABSTRACT: We report, for the first time, the design and fabrication of an electrochemical ion (E-ION) sensor for highly specific detection of hexavalent chromium (Cr(VI)). Unlike previously developed electrochemical Cr(VI) sensors, the sensing mechanism relies on the previously unexplored electrocatalytic reaction between Cr(VI) and surface-immobilized methylene blue (MB). The sensor is sensitive, specific, and selective enough to be used in a synthetic aquifer sample. Like many sensors of this class, it is also reagentless, reusable, and compatible with gold-plated screen-printed carbon electrodes. Despite the difference in the sensing mechanism, this E-ION Cr(VI) sensor possesses attributes similar to other MB-based electrochemical sensors, sensors with potential for real world applications.

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chemical detection methods, including anodic stripping voltammetry (ASV), have shown to be promising techniques for Cr(VI) detection. These methods are equally sensitive but require less expensive equipment. While numerous ASV-based Cr(VI) sensors have been reported in recently years, challenges in electrochemical detection of Cr species remain because of the different possible oxidation states. Most sensing strategies require the use of a chelating agent for Cr speciation.11−14 There is a clear unmet need for sensor technologies capable of sensitive, specific, and selective detection and quantification of Cr(VI). In this study, we exploit the high oxidation state of Cr(VI) in the fabrication of a reagentless and reusable electrochemical Cr(VI) sensor that is also specific and selective. The signaling mechanism relies on the electrocatalytic reaction between Cr(VI) and surface-immobilized methylene blue (MB). MB is first reduced at the electrode to form leucomethylene blue (LMB), the reduced form of MB. LMB proceeds to catalyze the reduction of freely diffusing Cr(VI) to form Cr(III) and regenerate MB in the process (Scheme 1).15,16 The reaction stoichiometry is different from the well-characterized electro-

any metal ions that are introduced into natural waters as waste from industrial processes are toxic to humans, thus the release of these substances has to be regulated and carefully monitored.1 Hexavalent chromium (Cr(VI)) is one of the most toxic metal contaminants, especially when compared to trivalent chromium (Cr(III)). Unlike Cr(III), Cr(VI) is more mobile and is often found in potable waters.2 In addition to naturally occurring Cr(VI), various industries, including metal plating and stainless steel production industries, utilize chromic acid or other forms of Cr(VI).3 This heavy metal contaminant is also found in inks, pigments, fungicides, and paints.4,5 Exposure to Cr(VI) in an aerosol form has been shown to cause chronic ulcers, dermatitis, and even lung cancer.5 While the carcinogenic effects of Cr(VI) consumption on humans have not been studied extensively, a previous study has shown that mice and rats contracted malignant tumors in their small intestines and mouths after consuming different doses of Cr(VI).6,7 Owing to the malignancy associated with exposure to Cr(VI), a wide range of analytical techniques capable of sensitive detection of Cr(VI) have since been developed. Analysis of Cr(VI) is most commonly performed using atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and fluorescence spectroscopy.8−10 Many of these methods are sensitive and accurate, but they are also costly and require sophisticated instrumentations. Electro© XXXX American Chemical Society

Received: January 15, 2015 Accepted: February 11, 2015

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Analytical Chemistry Scheme 1. Design and Signaling Mechanism of the E-ION Sensor for Detection of Cr(VI)

catalytic reaction between MB and ferricyanide;17,18 in this case, three equivalents of LMB are required to react with two equivalents of Cr(VI).16 Although this electrochemical ion (E-ION) sensor design does not require the use of a biorecognition element, the use of surface-immobilized MB enables this sensor to possess qualities similar to the well-characterized electrochemical DNA (EDNA) sensors, which include good sensor specificity, selectivity, and reusability.19−22 Like the E-DNA sensors, this E-ION sensor can also be used with gold-plated screen-printed carbon electrodes (SPEs).23 These attributes distinguish the current Cr(VI) sensor from previously developed Cr(VI) sensors. The sensor fabrication protocol is similar to that used with the E-DNA sensors. In brief, we placed an electrochemically cleaned gold electrode in a solution containing 30 μM MBterminated C6 alkanethiol probe (MB-P) for 1 h (Figure S1 in the Supporting Information). The electrode was subsequently passivated via an overnight incubation in 2 mM 6-mercapto-1hexanol. Cyclic voltammetry (CV) was first used to characterize the sensor. In the absence of the target, we observed a set of redox peaks at a half-wave potential (E1/2) of ∼−0.12 V (vs Ag/ AgCl), confirming successful immobilization of MB-P on the electrode surface (Figure 1A). The lack of a large hysteresis (peak-to-peak separation) implies efficient electron transfer between the substrate electrode and the surface-immobilized MB. The shift of the E1/2 to a more positive value is due to the use of an acidic buffer (pH 4.5 acetate buffer) in this study. This is expected given that MB is protonated upon reduction; this reaction is thus dependent on the solution pH.24 A more acidic pH is necessary for optimal detection of Cr(VI) via this electrocatalytic route.15,16 This pH, however, is significantly higher than that used in a previous study (∼pH 2).16 To verify the sensor’s specificity for Cr(VI), we first interrogated the sensor with 50 μM Cr(III). No change in the CV scan was evident; this is expected since Cr(III) is incapable of partaking in the electrocatalytic reaction (Figure 1A). Upon addition of 5 μM Cr(VI), we observed a simultaneous increase and decrease in the MB reduction and oxidation peak currents, respectively. This observation is consistent with our proposed sensing mechanism, in which the electrochemically generated LMB reacts with Cr(VI) to form Cr(III) and regenerate MB. The regenerated MB then participates in the electrocatalytic cycle, resulting in the increase in the reduction peak current. This sensor has clearly demonstrated its ability to discriminate between Cr(VI) and Cr(III), despite Cr(III) being 10 times higher in concentration. Furthermore, the detection is relatively rapid; all CV scans were collected 5 min after the addition of the target, and no further change in the current was recorded at longer time intervals.

Figure 1. CV scans of the E-ION sensor fabricated on a gold disk electrode in the absence, presence of 50 μM Cr(III), 5 μM Cr(VI) and after sensor regeneration (A). Also shown are CV scans of the sensor fabricated on a gold-plated SPE and interrogated under the same experimental conditions. The roughness factor (real area/geometric area) of this electrode was ∼1.5 (B). All scans were collected in a pH 4.5 acetate buffer at a scan rate of 10 mV s−1.

Like most sensors of this class, the E-ION sensor is also regenerable. Sensor regeneration can be achieved by simply rinsing the electrode with deionized water for ∼30 s. The CV scan recorded after sensor regeneration was close to identical to the scan obtained prior to the addition of Cr(VI). The sensor was subsequently interrogated with 50 μM Cr(III) and 5 μM Cr(VI) (Figure S2 in the Supporting Information). The responses were comparable to that observed with a freshly prepared sensor, verifying the sensor’s reusability. It is noteworthy that the sensor was stable for ∼8 h; no signs of monolayer degradation were detected. Direct reduction of Cr(VI) onto the sensor surface was not detected under the current experimental conditions.25 ASV and oxidative desorption of the monolayer were used to confirm the lack of electrodeposited Cr on the electrode surface.26,27 Although gold disk electrodes are the most commonly used electrodes for this class of electrochemical sensors, we have successfully fabricated these sensors on gold-plated SPEs, an advancement that enables these sensors to move from the laboratory into real world use.23 CV scans of the E-ION sensor fabricated on a gold-plated SPE are shown in Figure 1B. The sensor showed minimal change in both the reduction and oxidation currents in the present of 50 μM Cr(III), whereas a measurable increase in the MB reduction peak current was evident upon the addition of 5 μM Cr(VI). The MB oxidation peak current was slightly larger than that observed with the sensor fabricated on a gold disk electrode; this could be B

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Analytical Chemistry

× 1012 molecules cm−2. Since the sensor’s signaling capacity is inherently dependent on the probe coverage, we systematically evaluated the effect of probe coverage on the sensor’s response to its target. Like most sensors of this class, the probe coverage can be readily tuned by simply changing the concentration of thiolated probe used in the probe immobilization step.19−22 In addition to 30 μM, two other concentrations of MB-P (60 μM and 15 μM) were used in this study. For this sensor design, a higher probe coverage (i.e., more available redox sites) can potentially improve the overall sensor performance; our results, however, suggest otherwise. Sensors with a high probe coverage (e.g., 2.3 (±0.6) × 1013 molecules cm−2) showed lower % SE, implying the sensor’s response is not only dependent on the number of probes but also the distribution of the probes on the sensor surface (Figure S3A in the Supporting Information). The broad redox peaks also suggest the lack of homogeneity in the distribution of MB-P on the sensor surface (Figure S3B in the Supporting Information). Although attempts have been made to improve monolayer homogeneity in sensors, clustering of probes in self-assembled monolayer-based sensors cannot be entirely eliminated.29 The use of a lower probe coverage can in theory alleviate this clustering effect; while a higher % SE was observed using sensors with a lower probe coverage (e.g., 9.2 (±1.5) × 1011 molecules cm−2), the LOD remained unchanged (Figure S4A in the Supporting Information). The main disadvantage of using a sensor with a low probe coverage is the small MB peaks, which renders quantitative determination of the peak height/ current or integrated charge more challenging (Figure S4B in the Supporting Information). A medium probe coverage obtained using 30 μM MB-P was found to be ideal for this sensor; this experimental protocol was thus used for the rest of the study. In addition to CV, chronoamperometry (CA) was also used to evaluate the sensor’s sensitivity. Shown in Figure S5A in the Supporting Information is the sensor’s response to Cr(VI) when interrogated using CA at an applied potential of −0.25 V (vs Ag/AgCl). The current measured after 10 s was plotted as a function of target concentration. Figure S5B in the Supporting Information shows the chronoamperograms recorded in the absence and presence of different concentrations of Cr(VI). A linear increase in the current was observed between 500 nM and 10 μM Cr(VI) (Figure S5A inset in the Supporting Information). Concentrations higher than 100 μM did not result in higher % SE. The LOD was found to be 500 nM, higher than that obtained using CV. This could be due to a larger current fluctuation (i.e., noise) in CA, when compared to CV, in the absence of the target. The use of a different analysis time was also explored; however, the sensor’s response did not improve and the LOD remained the same. High sensor specificity is required for all real world sensing applications; thus, in addition to Cr(III), we analyzed the sensor’s response to 13 other potentially interfering metal ions. Shown in Figure 3A are the % SE recorded in the presence of each of the 14 metal ions. The sensor did not respond to most ions other than Fe(III). The response to Fe(III) varied widely, as reflected by a large standard deviation. Nevertheless, the change in the MB current was much lower than that recorded in the presence of Cr(VI). It is worth mentioning that this sensor functions well under ambient conditions; none of the experiments were conducted in an inert atmosphere. Furthermore, the presence of oxidants such as hydrogen peroxide did not interfere with Cr(VI) analysis (Figure S6 in

attributed to the surface roughness of the electrode. It is likely that there are MB-P immobilized on parts of the electrode that are not accessible to Cr(VI) and thus cannot participate in the electrocatalytic process.23 Regeneration of the sensor was successful, only minor differences between the CV scans obtained before target interrogation and after sensor regeneration were evident. In the detection of highly toxic metal ions such as Cr(VI), sensitivity becomes one of the most important sensor properties. To determine the limit of detection (LOD) for Cr(VI), we obtained two dose−response curves by analyzing the change in the reduction peak current and the integrated charge before and after the addition of different concentrations of Cr(VI) (Figure 2). Independent of the analysis method, a

Figure 2. Dose−response curves for Cr(VI) obtained by analyzing both the MB reduction peak current and integrated charge in the CV scans. The inset shows the sensor’s response to lower concentrations of Cr(VI). The data are representative of three independent experiments. 30 μM MB-P was used in the fabrication of these sensors (A). CV scans in the absence and presence of 0.05, 0.1, 0.5, 1, 5, 10, and 50 μM Cr(VI) (B).

linear increase in the % signal enhancement (% SE) was observed between 100 nM and 50 μM Cr(VI). Concentrations higher than 50 μM did not result in higher % SE. Despite the slight difference between the slopes of the two dose−response curves, the LOD was 100 nM in both cases. This LOD is significantly lower than the acceptable total Cr concentration (100 ppb or 1.9 μM) according to the United States Environmental Protection Agency standard.28 It is worth noting that this LOD was determined from sensors fabricated using 30 μM MB-P in the probe immobilization step and with a probe coverage of 4.3 (±0.6) C

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of hydrogen peroxide and Cr(VI). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (Grant CHE-0955439) and Nebraska EPSCoR (Grant EPS1004094).

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Figure 3. %SE recorded in the presence of 50 μM of Ca(II), Mg(II), Li(I), K(I), Zn(II), Ni(II), Cs(I), Co(II), Cr(III), Mn(II), Cd(II), Pb(II), Fe(III), 5 μM Hg(II) and Cr(VI). The data are representative of three independent experiments (A). CV scans of the sensor in the absence and presence of 5 μM Cr(VI) in a 50% synthetic aquifer sample. The synthetic aquifer sample was formulated according to the South Dakota Minnelusa Aquifer (B).

the Supporting Information). To determine the sensor’s performance in a real world sample, we interrogated the sensor in a 50% synthetic aquifer sample.30 As shown in Figure 3B, despite the shift in the redox potential which is likely due to the change in pH, the sensor did not respond to the various ions in the aquifer sample; addition of 5 μM Cr(VI) resulted in % SE similar to that seen in a pure buffer (Figure 1A). In conclusion, we have developed a new approach for realtime detection of Cr(VI) without interference from Cr(III) and a wide range of other metal ions. The sensing strategy exploits the previously unexplored electrocatalytic reaction between Cr(VI) and surface-immobilized MB. This sensor has proven to be highly sensitive, specific, and selective. The use of low-cost, paper-based electrodes further improves the applicability of this sensor for real world analysis of environmental and food samples.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

Experimental conditions; structure of MB-P; sensor interrogation-regeneration plot; dose−response curves using CV and corresponding voltammograms (60 μM MB-P); dose− response curves using CV and corresponding voltammograms (15 μM MB-P); dose−response curves using CA and corresponding chronoamperograms; CV scans in the presence D

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Analytical Chemistry (30) Williamson, J. E.; Carter, J. M. Water-Quality Characteristics in the Black Hills Area, South Dakota, U.S. Geological Survey, WaterResources Investigations Report 01-4194; 2001.

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