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Sensors and Actuators B 129 (2008) 896–902

Development of a method for total plasma thiols measurement using a disposable screen-printed carbon electrode coupled with a MnO2 reactor Chun-Yen Liao, Jyh-Myng Zen ∗ Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan Received 28 May 2007; received in revised form 18 September 2007; accepted 1 October 2007 Available online 10 October 2007

Abstract We report here an electrochemical approach for total plasma thiols measurement using a disposable screen-printed carbon electrode coupled with a MnO2 reactor. In this approach, catechol is used as an electrochemical indicator to monitor the nucleophilic addition of thiols to the o-quinone. A reactor containing MnO2 particles is effectively combined into the flow injection analysis (FIA) system not only to facilitate the oxidation of catechol but also to eliminate the interference from ascorbic acid. At a low operating potential of −0.1 V versus Ag/AgCl, the interference by electroactive species (e.g., uric acid) and physiological constituents (e.g., lysine) can also be avoided. The analytical parameters in FIA for the determination of thiols (cysteine, homocysteine and glutathione) have been assessed. The proposed approach is finally validated through its application to the analysis of total thiols in human plasma. © 2007 Elsevier B.V. All rights reserved. Keywords: Screen-printed electrode; Disposable; Thiols; Plasma

1. Introduction Homocysteine, glutathione, cysteinylglycine and cysteine are the most common plasma low-molecular-mass aminothiols involved in important functions in metabolism and homeostasis. The quantification of thiols in biological fluids is therefore useful for the diagnosis of some serious illness [1–3]. So far, among the reported methods, such as amino acid analyzer as well as radioenzymatic and chromatographic methods [4–8], the combination of separation techniques with electrochemical detection is particularly attractive because of the electrochemical activity of thiols [9–17]. Successful exploitation of such an approach, however, is dependent upon the availability of techniques that can provide fast quantitative measurements of the various biomarker concentrations. Electrochemical techniques would appear to be particularly suited to such a task given the flexibility of the redox chemistry that characterizes most organo-sulphur compounds [16,17]. Electrochemical methods have been proved useful for such a purpose also because of the ease in automation and high sensitivity [18–21]. However, such

∗

Corresponding author. E-mail address: [email protected] (J.-M. Zen).

0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.10.004

methods have been limited mainly by the sluggish electrochemical process of thiols at common electrodes, e.g., glassy carbon and gold electrodes [22,23]. Chemically modified electrode can partially solve the problem, e.g., an electrode constructed by entrapment of the coenzyme pyrroloquinoline quinone (PQQ) into a polymer matrix to serve as an efficient biocatalyst to mediate the oxidation of thiols can operate at a substantially reduced overpotential relative to an unmodified electrode [24]. Although the electrochemical responses of these compounds have been continuously improved, the development of an electrochemical method for sensitive and selective determination of thiols still remains very challenging. We report here a method for total plasma thiols measurement using a disposable screen-printed carbon electrode (designated as SPE) coupled with a MnO2 reactor by flow injection analysis (FIA). It is well known that catechol can be electrochemically converted to o-quinone and readily undergoes reaction with thiols possessing sulphydryl groups [25]. In fact such a thiol/oquinone reaction is responsible for the electrocatalysis towards the thiols [26–28]. Unfortunately, in the applied oxidative potential, the electrodes were found to be particularly susceptible to fouling [29]. The main purpose of the proposed approach is therefore to develop a method to avoid this electrode-fouling phenomenon. The main idea is by setting up the oxidative reac-

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tion of catechol at a MnO2 reactor and the following nucleophilic addition of thiols to the o-quinone can be monitored electrochemically at a disposable SPE. To monitor the inhibited current of the reductive signal of o-quinone can thus be exploited as a means of quantifying the concentration of the thiols. Note that the MnO2 reactor was effectively combined into the system not only to facilitate the oxidation of catechol but also to eliminate the interference from ascorbic acid and others by detecting at a low detection potential of −0.1 V versus Ag/AgCl. Precise thiols determination can consequently be easily performed by FIA in aerobic condition. We believe that the proposed method is a good alternative for the routine sensing of total thiols in human plasma due to its simplicity of preparation, low susceptibility to electrode fouling, low detection limit and insensitivity to interference from ascorbic acid and other electroactive easily oxidizable bio-molecules. 2. Experimental 2.1. Chemicals and reagents All reagents were of the highest grade available and used without further purification. All solutions and subsequent dilutions were prepared daily using deionized water from a Millipore-Q purification system with a resistivity of 18 M cm−1 and were refrigerated when not in use. Stock solutions of 0.01 M catechol, cysteine, homocysteine and glutathione were prepared through the dissolution of the appropriate salt in deionized water and generally used within 1 h. The manganese dioxide powder packing in the reactor was obtained from Riedel-de Ha¨en. Phosphate buffer solution (PBS) (0.1 M, pH 7.0) was used throughout with the exception of those experiments engaged in pH studies. 2.2. Apparatus and measurements Cyclic voltammetric and chronoamperometric experiments were preformed with a CHI 900 electrochemical workstation (Austin, TX, USA). The three-electrode system was constructed from an Ag/AgCl (3 M KCl) reference electrode, a platinum auxiliary electrode and a SPE-based working electrode. Disposable SPEs were purchased from Zensor R&D (Taichung, Taiwan). The commercial available Zensor SF-100 flow injection electrochemical cell is fabricated by using a high performance engineering polymer (i.e., polyoxymethylene (acetal) copolymer) that can resist impact, fatigue and chemicals with high strength, stiffness and low moisture absorption [30]. The geometric area is 0.196 cm2 for SPE. The FIA system consisted of a Cole-Parmer microprocessor pump drive, a Rheodyne model 7125-sample injection valve (20 ␮l loop) with interconnecting Teflon tube and a Zensor SF-100 thin-layer detecting electrochemical cell specifically designed for SPE [30]. The SPE was first washed thoroughly with deionized water before placing into the electrochemical flow cell. The electrode was equilibrated in a carrier solution of PBS (0.1 M, pH 7) by cyclic voltammetry (−0.8 to +1.0 V) until the curve current became constant. The reactor was prepared by packing 0.4 g MnO2 powder in a tube

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and placed between the sample injector and the electrochemical cell in the FIA system. The carrier solution containing catechol flows through the reactor and the reductive current of o-quinone can then be collected. The FIA responses by thiols that inhibited the reductive current of o-quinone were used for the analytical purpose. 3. Results and discussion 3.1. Electrochemical characterization To confirm the reaction mechanism that catechol can be electrochemically converted to o-quinone and readily undergoes reaction with thiols possessing sulphydryl groups, cyclic voltammetry for 60 ␮M catechol in the presence/absence of cysteine was first studied at a SPE in pH 7 PBS. As can be seen in Fig. 1, the oxidation of catechol with a well-defined peak at +0.43 V together with the corresponding reduction peak at +0.04 V was observed. Most importantly the addition of cysteine leads to an increase in the oxidation peak current and is accompanied with a decrease in the reduction peak current. The increase in the oxidation peak current is attributed to the oxidation of catechol–cysteine adducts and the successive decrease in the reduction peak can be ascribed to the fact that increasing concentrations of cysteine serve to scavenge the oxidized form of catechol such that on the reverse sweep there is little available for electroreduction. Overall, the typical voltammetric features observed are as expected and follow the reaction pathway as outlined in Scheme 1. Since the method is aimed at total plasma thiols measurement, the influence of pH on the reaction is next assessed by studying the electrode response to cysteine between pH 1.5 and 9. Fig. 2 compares the cyclic voltammograms of 150 ␮M catechol in the absence and presence of 50 ␮M cysteine at pH 1.54, 6.70 and 9.15, respectively. As can be seen, the redox potential is pH-dependent and the responses decrease steadily as the acidity of the solution increases from pH 6.70 to 1.54.

Fig. 1. Cyclic voltammograms of 60 ␮M catechol (solid line) obtained at a bare SPE by scanning positively at −0.2 V in pH 7.0 PBS towards increasing concentrations of cysteine (20 and 40 ␮M, dashed line). Scan rate, 20 mV/s.

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Scheme 1.

Fig. 2. Cyclic voltammograms of 150 ␮M catechol obtained at a bare SPE in pH 1.54, 6.70 and 9.15 in the absence (dashed line) and presence of 50 ␮M cysteine (solid line). Scan rate, 20 mV/s.

This can be attributed to the fact that as the solution pH is lowered, the thiol functionality is increasingly protonated (cysteine, pKa = 8.4) and hence the nucleophilic character of the thiol moiety diminishes. Increasing of the solution pH clearly improves the response but an operational limit is reached once neutral conditions prevail. Alkaline solution (e.g., pH 9.15)

severely reduces the response as the increased presence of nucleophilic hydroxyl ions (and amine groups where complex physiological media are employed) compete with the less prevalent thiol [29]. Overall, a neutral pH seems to be acceptable for the purpose of study and is therefore used in the subsequent studies.

Fig. 3. Consecutive cyclic voltammograms of 200 ␮M catechol obtained at a bare SPE by scanning negatively at +0.15 V without (A) and with (B) the addition of 0.2 g MnO2 powder in solution. Scan rate, 20 mV/s.

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3.2. The utility of MnO2 reactor As mentioned earlier catechol can be electrochemically converted to o-quinone; yet unfortunately, the electrodes were found to be particularly susceptible to fouling in the applied oxidative potential [29]. It is expected that MnO2 is a strong oxidant that can oxidize catechol to o-quinone and thus, by using a MnO2 reactor to oxidize catechol, this electrode-fouling phenomenon should be avoided. To evaluate the effectiveness of the MnO2 reactor, scan segment experiments for the reaction of catechol in pH 7 PBS were performed. As shown in Fig. 3A, the reduction peak of catechol cannot be observed in the first scan segment as the cyclic voltammetric scan negatively from 0.15 V. In other

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words, catechol must be oxidized first and then the reduction of o-quinone can occur. This is not the case after the addition of MnO2 powder. As shown in Fig. 3B, catechol is chemically oxidized to o-quinone by MnO2 and hence the reduction peak can be observed in the first scan. The effectiveness of the MnO2 reactor is confirmed from this study and can thus be combined into the FIA system to facilitate the oxidation of catechol. The other advantage of using the MnO2 reactor is for the removing of interference as the insoluble MnO2 powder can oxidize ascorbic acid efficiently. Fig. 4A demonstrates the interference-removing efficiency of MnO2 toward ascorbic acid. As can be seen, the electrochemical signal from ascorbic acid was found to completely disappear after the addition of MnO2

Fig. 4. (A) Cyclic voltammograms of 200 ␮M ascorbic acid obtained at a bare SPE without (solid line) and with (dashed line) the addition of 0.2 g MnO2 powder in solution. (B) Cyclic voltammograms of 100 ␮M catechol in pH 7.0 PBS towards increasing concentrations of uric acid (100, 200 and 300 ␮M). Scan rate, 20 mV/s. (C) The FIA responses of 50 ␮M catechol in carrier solution (pH 7.0 PBS) with injecting 50 ␮M glutathione and other interferants (in the absence of glutathione) of ascorbic acid, uric acid and lysine at a detection potential of −0.1 V and a flow rate of 0.8 ml/min. Injected sample volume, 20 ␮l.

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Fig. 5. The optimization conditions of (A) packing amount of MnO2 , (B) detection potential and (C) flow rate on the FIA responses of 100 ␮M cysteine.

into the solution. The interference form uric acid can be further demonstrated by the following experiment. Fig. 4B shows the cyclic voltammograms of 100 ␮M catechol in pH 7.0 PBS towards increasing concentrations of uric acid (100, 200 and 300 ␮M). As can be seen, the oxidation peaks were found to increase with the increase in concentration of uric acid. Whereas, the reduction peaks were virtually the same. In other words, the reductive reaction of o-quinone is indeed not affected by increasing the concentration of uric acid. Most importantly, since the amperometric signal of o-quinone can be collected at a low detection potential of ∼0.0 V, interference by other electroactive species can also be avoided in FIA. To confirm this, the effectiveness of the proposed system in the removal of interference was demonstrated by the following experiments. Fig. 4C shows the FIA responses of 50 ␮M glutathione and other interferants of electroactive species (e.g., ascorbic acid and uric acid) and physiological constituents (e.g., lysine). The determination of thiols by using the proposed system to get away from these interferants is confirmed in the study. Meanwhile the function of the MnO2 reactor for eliminating of interference from ascorbic acid and for oxidizing catechol to produce o-quinone was also validated in these experiments. 3.3. Parameters optimization in FIA Parameters that can affect the FIA performance of the proposed system for total plasma thiols measurement were thoroughly studied. In order to optimize the efficiency of reactor in FIA, the packing amount of MnO2 powder was first evaluated and the results obtained was shown in Fig. 5A. As can be seen, as the packing amount MnO2 was lower than 0.4 g, full oxidation of catechol cannot be achieved. Whereas, as the amount of MnO2 was higher than 0.4 g, the flow pathway was somewhat restricted and thus caused a large deviation in the detection signal. A packing amount of 0.4 g MnO2 was thus chosen in subsequent studies. The effect of detection potential and flow rate were next examined. As can be seen in Fig. 5B, the peak current

increases with the increase in potential negatively and remains constant after −0.1 V. This is as expected and matches with the cyclic voltammetric results as mentioned in the earlier section. Finally, the effect of flow rate in FIA was evaluated. As shown in Fig. 5C, the current signals were found to increase sharply with the increase in flow rate. Nevertheless as the flow rate was higher than 0.8 ml/min, much larger deviation in the detection signals was obtained. Overall, since the signal obtained from a flow rate of 0.8 ml/min showed a better sensitivity and repeatability, a flow rate of 0.8 ml/min was thus chosen for further analytical applications. Under these optimized FIA conditions, the precision of 12 repetitive determinations of 50 ␮M cysteine, homocysteine and glutathione were evaluated (data not shown). A relative standard deviation of ∼1% validates good reproducibility of proposed method in the detection of thiols. Fig. 6 shows the typical FIA responses of various concentrations of glutathione and the obtained calibration plot. The positive background current can be attributed to the reductive reaction of o-quinone that was produced by 50 ␮M catechol in the carrier solution flowing through the reactor. The extended signal current linearity region (5–80 ␮M) versus concentration with a slope of 0.01 ␮A/␮M and a detection limit of 0.47 ␮M (S/N = 3) were observed. Note that the sensitivity towards homocysteine and glutathione are significantly less than that of cysteine (detection limit = 0.10 ␮M). A more quantitative evaluation of the performance of the assay towards each thiol is presented in Table 1.

Table 1 Calibration characteristics of cysteine, homocysteine and glutathione using the proposed system [Catechol] (␮M)

Thiols

Linear range (␮M)

R

LOD (␮M)

50 50 50

Cysteine Homocysteine Glutathione

5–50 5–80 5–80

0.997 0.998 0.999

0.10 0.25 0.47

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Fig. 6. (A) FIA responses of 50 ␮M catechol in carrier solution (pH 7.0 PBS) with injecting different concentrations of glutathione at a detection potential of −0.1 V and a flow rate of 0.8 ml/min. Injected sample volume, 20 ␮l. (B) The calibration curve of glutathione.

Table 2 Total plasma thiols measurement by the proposed system and standard method

4. Conclusions

Real sample

This is the first use of disposable screen-printed carbon electrodes for the electroanalysis of total plasma thiols. Catechol employed as a derivative reagent for thiols determination in complex biological media has been demonstrated. The reaction has been shown to be specific for sulphydryl thiols (RSH) with an analytically relevant detection range that is comparable to the reported results [32,33]. The MnO2 reactor was introduced to the FIA system for the oxidation of the catechol and the elimination of the interference from ascorbic acid. The response is sufficiently sensitive and selective to deal with the complex matrix provided by human plasma and compares well with the traditional spectroscopic protocol. The results clearly demonstrate the validity of the approach for the analysis of total plasma thiols.

The proposed method Ellmans reagent (UV–vis)

Total plasma thiols (mM) #1

#2

#3

0.54 0.57

0.47 0.50

0.56 0.62

The calibration characteristics of all the three thiols studied show satisfied results. 3.4. Real sample application Finally, the proposed method was applied to real sample application by detecting the amount of thiols in human plasma. The experimental conditions, as described in the previous section, were applied to determine the quantity of thiols present in real samples. The obtained results are summarized in Table 2. As can be seen, the amounts of total thiols measured for the three real samples are in good agreement with those detected by traditional spectroscopic protocol of Ellmans reagent. The analytical viability of the approach to the analysis of real samples was thus assessed through the determination of total plasma thiols. Moreover the concentration of total plasma thiols was also found to be in agreement with previous studies examining the influence of reactive oxygen species on the concentration of thiol antioxidants [31] and serves to corroborate the viability of the approach. Overall the results verify the use of simple and easy electrochemical method for fast and reliable measurement of total plasma thiols.

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Biographies Chun-Yen Liao is currently pursuing his doctoral studies under the guidance of Prof. Jyh-Myng Zen. His interests are in polymer modified metal oxide electrodes, electrocatalysis, and chemical sensors. Jyh-Myng Zen was born in Taipei, Taiwan in 1957. He obtained BS degree in chemistry from National Tsing Hua University, Taiwan, in 1980. He served military service by duty in 1980–1982. He studied electrochemistry with Larry R. Faulkner at University of Illinois, Urbana-Champaign and graduated with a PhD in 1988. For the next three years, he was a postdoctoral researcher with Allen J. Bard and John B. Goodenough first at Chemistry Department and later at Center for Material Science and Engineering in University of Texas–Austin. He is currently a professor at Department of Chemistry, National Chung-Hsing University (NCHU), Taiwan. His research interests include chemically-modified electrodes, electrocatalysis, physical electrochemistry, and chemical sensors.