Enantioselective recognition of chiral mandelic acid in the presence of Zn(II) ions by l -cysteine-modified electrode

Sensors and Actuators B 155 (2011) 140–144

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Enantioselective recognition of chiral mandelic acid in the presence of Zn(II) ions by l-cysteine-modified electrode Yingzi Fu ∗ , Lilan Wang, Qiao Chen, Juan Zhou Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, No. 2 Tiansheng Road, Chongqing 400715, PR China

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Article history: Received 14 July 2010 Received in revised form 16 November 2010 Accepted 22 November 2010 Available online 26 November 2010 Keyword: Electrochemical investigation Enantioselective recognition Mandelic acid Zn(II) ions l-Cysteine-modified electrode

a b s t r a c t An obviously enantioselective strategy for the recognition of mandelic acid (MA) enantiomers in the presence of Zn(II) ions on a l-cysteine (l-Cys) self-assembled gold electrode is described. The high recognition of MA was evaluated via electrochemical impedance spectroscopy and cyclic voltammetry. After the modified electrode interacted with R- or S-MA solution containing Zn(II) ions for 10 min, larger electrochemical response signals were observed for R-MA. Time dependencies of the enantioselective interaction for the modified electrode with the solitary Zn(II) solution and MA enantiomers solutions containing Zn(II) were also investigated. The results showed that the enantioselective recognition was caused by the selective formation of Zn complex with l-Cys and MA enantiomers. In addition, the enantiomeric composition of R- and S-MA enantiomer mixtures could be monitored by measuring the current responses of the sample. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Chiral recognition is important in pharmaceutical sciences, drug screening and biological processes because of the high selectivity of chiral molecular species in nature and life [1–6]. Chiral mandelic acid (MA) plays a significant role in the pharmaceutical synthetic industry. For example, R-mandelic acid (R-MA) is used not only as a key intermediate for semisynthetic penicillin and cephalosporin, but also as a chiral resolving agent and chiral synthon for the synthesis of anti-tumor agents [7]. Developing practical, rapid and available methods for the chiral recognition of MA is valuable and fascinating. Various discrimination techniques about MA have been reported, such as diastereomeric crystallization [8,9], high-performance liquid chromatography (HPLC) [10], quartz crystal microbalance (QCM) [11], fluorescence detection [12] and electrochemical detection [13]. And with the advantages of simple operation, rapid detection and low cost, chiral electrochemical sensor have potential applications in pharmaceutical systems and clinic diagnoses [14–17]. Some amino acids were investigated as the selector in chiral electrochemical recognition [11,18]. Owing to amino acids can form metal complexes with metal ions, such as Cu(II), Zn(II) [19,20], the principle of chiral ligand exchange has been used in

∗ Corresponding author. Tel.: +86 023 68252360; fax: +86 023 68253195. E-mail address: [email protected] (Y. Fu). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.11.038

the recognition and separation of amino acids and their derivatives, especially in chromatography analysis [21–23]. Here we describe a new strategy for highly recognizing target MA enantiomers. The recognition is based on the enantioselective forming metal complexes with MA enantiomers (a chiral ␣-hydroxy carboxylic acid, which can use as a chiral ligand in the synthesis of chiral drugs [24,25]) and l-cysteine in the presence of Zn(II) ions. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to monitor the enantioselectivity. These stereospecific responses were sensed quickly according to the remarkably different changes in current and impedance responses.

2. Experimental 2.1. Reagent and apparatus l-Cysteine (l-Cys), R- and S-mandelic acid (R- and S-MA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). K3 [Fe(CN)6 ], K4 [Fe(CN)6 ], Zn(OOCCH3 )2 and other chemicals were analytical grade. Double distilled water was used throughout this study. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements were carried out on a CHI 660D electrochemistry workstation (Shanghai Chenhua Instruments Co., China).

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2.3. Experimental measurements Electrochemical experiments were performed in a threeelectrode electrochemical cell containing a working electrode (the modified electrode or bare electrode), a platinum wire auxiliary electrode and a saturated calomel reference electrode (SCE). After the l-Cys-Au electrodes were immersed in 1 ml S-MA or R-MA solution (containing 0.05 mM Zn(OOCCH3 )2 , which denoted SMA + Zn(II) or R-MA + Zn(II), pH 5.5) for a certain time, cleaned carefully with water, then recorded the response signals in 5 mM [Fe(CN)6 ]4−/3− solution via CV or EIS techniques. The frequency range of EIS measurements from 0.1 to 105 Hz in a given open circuit voltage, amplitude was 0.22 V. The difference of reduction peak current (I) was given by the following equation: I = I0 − I1 , where I0 and I1 were the reduction peak currents before and after the interaction with MA + Zn(II), respectively. 3. Results and discussion 3.1. Determination of the surface roughness The surface roughness factor (Rf ) of the gold electrode plays an important role in the reproducible formation of high-quality self-assembled monolayer on gold electrode [27,28]. Rf is the ratio of real surface area, Areal and geometrical surface area, Ageom (Rf = Areal /Ageom ). Before immersed in the l-Cys solution, the surface roughness of the pretreated electrode was determined by CV using 5 mM [Fe(CN)6 ]4−/3− (0.1 M KCl) probe at different scan rates as the redox according to the Randles–Sevcik equation [29,30] IP = 2.69 × 105 AD1/2 n3/2 1/2 c0 where IP represents the peak current, n and c0 refer to the transferring electron number and the concentration of the ferricyanide, D is the diffusion coefficient of the molecule in solution,  is the scan rate. From the slope of the IP ∼ 1/2 relation, the average value of the active surface area (average of 3 measurements) for the pretreated gold electrode was 0.0895 cm2 , and Rf was 1.27. 3.2. Characteristics of the l-cysteine-modified electrode Compared the cyclic voltammograms (CVs) of the l-Cys-Au with bare gold electrode, a little decrease of peak current and minute migration of peak potential were observed. Based on the electrical charge associated with the reductive desorption of l-Cys by CV scanned in 0.5 M KOH, the molecular density of l-Cys on the gold electrode could be calculated as 9.10 × 10−10 mol cm−2 according to the literature [31].

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First, the gold electrodes (˚ = 3 mm) were polished on microcloth with 1.0, 0.3 and 0.05 ␮m alumina slurries, and sonicated sequentially in ethanol and water for 5 min each. Subsequently, the electrodes were treated with electrochemical method through three steps [26], (1) applied a positive potential of 2 V to the electrodes for 5 s, and followed by a negative potential of −0.35 V for 10 s, (2) scanned 10 cycles of CV between −0.3 and +1.55 V in 0.5 M H2 SO4 solution at a scan rate of 1 V s−1 , (3) run 10 cycles of CV again in a fresh 0.5 M H2 SO4 solution with the same potential range at 0.1 V s−1 , and rinsed with a copious amount of water. At last, the pretreated electrodes were immediately immersed in the 10 mM l-Cys solution for 12 h at room temperature, and the l-Cys modified gold electrodes (l-Cys-Au) were constructed successfully.

c -40

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Zre / Ohm Fig. 1. The electrochemical impedance spectroscopy (EIS) of different electrodes in 5 mM [Fe(CN)6 ]4−/3− solution: (a) l-Cys-Au and after l-Cys-Au interacted with (b) 1 ml S-MA + Zn(II) or (c) 1 ml R-MA + Zn(II) solution for 10 min. The concentration of MA was 10 mM, Zn(II) was 0.05 mM.

3.3. Enantioselective recognition of MA in the presence of Zn(II) EIS is considered to be an effective tool for probing the interface properties of surface-modified electrodes, and widely used to understand chemical transformations and processes associated with the conductive supports [32]. In EIS, the semicircle diameter in the impedance spectrum equals to the electron-transfer resistance, Ret [33,34]. This resistance controls the electron transfer kinetics of the redox-probe at electrode interface. Its value changes when different substances are adsorbed onto the electrode surface [35]. Fig. 1 showed the EIS of the enantioselective interaction of l-Cys-Au with MA in the presence of Zn(II). The Ret of l-Cys-Au (96.4 ) was a little larger than the Ret of bare electrode (85.7 ), indicating the formation of l-Cys monolayer slightly hindered the electron transfer of [Fe(CN)6 ]4−/3− on electrode surface. But the obvious increasing impedance were both exhibited in S-MA + Zn(II) and R-MA + Zn II) (Fig. 1b and c), confirming the occurrence of the interaction between l-Cys-Au and MA enantiomers solutions. More interesting, the impedance of R-MA + Zn(II) (Ret = 209 ) was larger than S-MA + Zn(II) (Ret = 128 ), hinting that the interaction between the modified electrode and R-MA + Zn(II) was stronger, and inducing a larger interfacial resistance on the electrode. It clarified that l-Cys-Au showed highly enantioselective recognition to MA enantiomers in the presence of Zn(II), especially to R-MA. In the meantime, CV detection was measured. As shown in Fig. 2A, after interacted with S- or R-MA + Zn(II) for 10 min, the peak current of the modified electrode decreased, suggesting that the interaction between l-Cys-Au and S- or R-MA + Zn(II) solution had been introduced a barrier on the electrode surface for electron transfer. And I of R-MA + Zn(II) (IR ) was larger than the I of S-MA + Zn(II) (IS ) too (curves b and c). The average difference of IR and IS was 32 ␮A, and the relative standard deviation (RSD) was 3.71%, n = 6. The result was consistent with EIS measurements. In order to explore the effect of Zn(II) in the chiral recognition, the interaction of l-Cys-Au with MA in the absence of Zn(II) was performed as a contrast experiment. From the curves b and c in Fig. 2B, the similar CVs and a little decrease of peak currents for R-MA and S-MA were observed after interacting for 10 min, but the changes of current were less than S-MA + Zn(II). The result emphasized that chiral recognition was not obtained for MA in the absence of Zn(II). Zn(II) was a crucial factor in the enantioselective recognition.

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Fig. 2. The CVs of different electrodes in 5 mM [Fe(CN)6 ]4−/3− solution. A (a) l-Cys-Au and after l-Cys-Au interacted with (b) 1 ml S-MA + Zn(II), (c) 1 ml R-MA + Zn(II) solution for 10 min; B (a) l-Cys-Au and after l-Cys-Au interacted with (b) 1 ml S-MA, (c) 1 ml R-MA solution for 10 min. The concentration of MA was 10 mM, Zn(II) was 0.05 mM.

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S-MA + Zn(II) and R-MA + Zn(II) solutions. Since the interaction between l-Cys-Au and Zn(II) ions was existed, the peak current in the solitary Zn(II) solution was decreased (Fig. 3a). The I of S-MA + Zn(II) (Fig. 3b) was slightly higher than the I of Zn(II) solution. For R-MA + Zn(II), the I kept highest in the range of 2–18 min, and leveled off after approximately 10 min although the current response was still enantioselective (Fig. 3c). According to the experimental results, the reasons of this enantioselectivity were discussed. The conceivable interactions between l-Cys-Au and MA + Zn(II) (or MA) solutions which leading to the decrease of current response are summarized as follows:

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Fig. 3. Time dependence of the enantioselective interaction for l-Cys-Au with (a) 1 ml solitary Zn(II), (b) 1 ml S-MA + Zn(II), (c) 1 ml R-MA + Zn(II). The concentration of MA and Zn(II) was 10 mM and 0.05 mM, respectively.

3.4. Time dependency of the enantioselective interaction Time dependencies of the enantioselective interaction were studied too. From Fig. 3, the decreased peak currents were observed in the modified electrodes after interacted with the solitary Zn(II),

(i) Electrostatic forces, H-bond, etc. between l-Cys and MA molecules [11]. (ii) The formation of Zn complex with l-Cys such as [Zn(II)(l-Cys)2 ] (Fig. 4a) [20]. (iii) The formation of Zn complex with l-Cys and MA such as [(lCys) Zn(II)(MA)] (Fig. 4b) (chiral ligand exchange interaction) [21,24,36–38]. In the MA enantiomers solutions without Zn(II), only the first kind of interaction between l-Cys-Au and MA is existed. The interacting force of this kind is too weak to induce a little decrease of peak current (b and c in Fig. 2B). In the solitary Zn(II) solution, the second kind of interaction is existed, which come from the form-

Fig. 4. Schematic of recognition interaction.

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suggested the potential application in quantitative chiral analysis [38].

a

Ya= 2.35 + 11.2 x Ra=0.9944

4. Conclusion

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(VR / VMA) ×100% Fig. 5. Current decrease of the l-Cys-Au with enantiomeric composition of RMA and S-MA in the presence of 0.05 mM Zn(II) at different concentrations: (a) 10 mM; (b) 1 mM. VMA was the total volume (1 ml) of enantiomeric composition (VMA = VR + VS ).

ing of [Zn(II)(l-Cys)2 ], produce a visible decrease of peak current (Fig. 4a). From Fig. 3b, the first and second kinds of interactions are performed in S-MA + Zn(II) solution (Here, S-MA is considered not to form Zn complex with l-Cys, Fig. 4b), resulting in a slightly larger current response than the solitary Zn(II). In R-MA + Zn(II) solution, three kinds of interactions are presented at the same time. The I of R-MA + Zn(II) is obviously higher than Zn(II) or S-MA + Zn(II), suggesting the third kind of interaction is predominant and resulting in the highest enantioselective recognition for MA. This enantioselective phenomenon may be caused by the different molecular configurations of MA enantiomers. The –COOH, –OH groups in MA enantiomers have connected to chiral carbon atoms, which possess different spatial arrangements. From the results, it can be presumed that the molecular configuration of R-MA match better with l-Cys, and induce a weaker steric hindrance during the formation of Zn complex ([(l-Cys)Zn(II)(R-MA)]). However, it is difficult for S-MA to form Zn complex with l-Cys because of the greater steric hindrance. In other words, the interaction groups are closer in the configuration of [(l-Cys)Zn(II)(R-MA)] complex than [(l-Cys)Zn(II)(S-MA)] complex, and the structure of [(l-Cys)Zn(II)(R-MA)] complex has higher stability (possessing lower energy) [36,37]. So the formation of [(l-Cys)Zn(II)(R-MA)] is easier and faster, and lead to the notable current decrease. In brief, the highly enantioselective recognition of chiral MA using l-Cys-Au is resulted from the enantioselective forming Zn complexes with l-Cys and MA enantiomers (Fig. 4b). 3.5. Application of the enantioselective sensor The l-Cys-Au was also used to determine the current responses of a series of solutions which were prepared by mixing R- and SMA at different fixed ratios containing 0.05 mM Zn(II). As shown in Fig. 5, the enantiomeric ratio of R- and S-MA mixtures could be determined from the calibration curves which showed good linearity. It could be seen no matter high or low concentration of MA, the changes of the peak current were nearly similar when the ratio of R-MA in MA mixtures was zero (only has S-MA + Zn(II)). At this ratio, the first and second kinds of interactions are presented. In comparison, the first interacting force is weak, and the second is major. It is worth pointing here that both concentrations of MA containing the same concentration of Zn(II) (0.05 mM). Thus, the nearly similar I for both concentrations of MA was reasonable at this ratio. This strategy was successfully used to detect the enantiomeric ratio of R- and S-MA mixtures, which

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Biographies Yingzi Fu is a vice-professor of chemistry in Southwest University, China, received her PhD degree in analytical chemistry from this university in 2006. Her main research interests are biosensors. Lilan Wang is a MS candidate in the College of Chemistry and Chemical Engineering of Southwest University, China. Her major interests are the development of electrochemical chiral sensor. Qiao Chen is a MS candidate in the College of Chemistry and Chemical Engineering of Southwest University, China, is interested in developing electrochemical devices for biosensors. Juan Zhou is a MS candidate in the College of Chemistry and Chemical Engineering of Southwest University, China, is interested in developing electrochemical devices for chiral analysis.