DNA-Inspired Electrochemical Recognition of Tryptophan Isomers by

Aug 29, 2015 - Inspired by the double helix structure of DNA, a simple enantioselective system based on chitosan (CS) was employed for electrochemical...
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DNA-Inspired Electrochemical Recognition of Tryptophan Isomers by Electrodeposited Chitosan and Sulfonated Chitosan Xiaogang Gu, Yongxin Tao,* Yan Pan, Linhong Deng, Liping Bao, and Yong Kong* Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology, School of Petrochemical Engineering, Changzhou University, Changzhou 213164, China ABSTRACT: Inspired by the double helix structure of DNA, a simple enantioselective system based on chitosan (CS) was employed for electrochemical enantiorecognition of tryptophan (Trp) isomers. The recognition mechanism was proposed from the supramolecular point of view, which was further verified by the recognition of Trp isomers with sulfonated CS (SCS). The SCS-based chiral system presented the ability of indicating the percentage of D-Trp in racemic mixture, extending future applications of the electrochemical chiral system based on natural polysaccharides.

fficient recognition of isomers by convenient techniques and simple materials is of great interest in medical science, pharmaceutics, and biochemistry.1,2 Among the reported technologies for chiral recognition, the electrochemical approach has drawn enormous attention because it overcomes the shortcomings such as the time-consuming and high cost associated with conventional chromatography.3−5 In particular, electroactive isomers are the most suitable candidates for chiral recognition via electrochemistry. In addition, although various artificial materials such as cyclophanes and crown ethers have been designed for recognition of isomers,2,6−9 the development of simple yet efficient materials for chiral recognition still remains an urgent task.10−12 DNA membranes are categorized as channel-type membranes, playing key roles in the chiral separation of racemic amino acids.13,14 Although its function is not fully understood, the presence of a double helix structure in the host molecules suggests a possible role in the recognition of chiral guest molecules from the supramolecular point of view. Chitosan (CS), a deacetylated derivative of chitin, is an optically active natural polysaccharide, and the excellent biocompatibility, biodegradability, and nontoxicity of CS make it an ideal candidate as biomaterial. More importantly, CS chains have an extended 2-fold helical conformation stabilized by O-3···O-5 hydrogen bonds,15,16 which is similar to that of DNA. Inspired by the chiral recognition with DNA membranes, we report on a simple and convenient chiral system based on CS electrodeposited on a glassy carbon electrode (GCE) for electrochemical recognition of tryptophan (Trp) isomers. This recognition may be attributed to the selective formation of Hbonds between CS and Trp isomers. The assumption is further verified by the recognition of Trp isomers with sulfonated CS (SCS), because the recognition efficiency is enhanced significantly due to the introduced alkyl sulfonate, which is beneficial to the formation of intermolecular H-bonds in the

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host−guest system. L-Trp preferentially permeates through the CS films to the electrode surface due to the higher binding ability of CS for D-Trp, and thus, the chiral recognition of Land D-Trp is achieved via monitoring the differences in oxidation peak potential (Ep) and oxidation peak current (Ip) signals in the voltammograms. More importantly, compared with previous research carried out with separated isomer solution,17−19 we present the ability of predicting the ratio of Dto L-Trp in racemic mixture, exhibiting the potential of the proposed polysaccharide-based chiral system in practical applications.

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EXPERIMENTAL SECTION Reagents and Apparatus. D-Tryptophan (D-Trp, 98%), Ltryptophan (L-Trp, 99%), and 1,3-propane sultone were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Potassium ferricyanide (K3Fe(CN)6) and potassium ferrocyanide (K4Fe(CN)6) were obtained from Shanghai Lingfeng Chemical Co., Ltd. (China). Chitosan (CS) with a deacetylation degree of 80∼95% was purchased from Sinopharm Chemical Reagent Co. (China). Electrochemical experiments were performed in a conventional three-electrode cell connected to a CHI-660D electrochemical workstation (CH Instruments, Inc., China). Working electrode was a glassy carbon electrode (GCE, 3 mm in diameter) modified with CS or sulfonated CS (SCS). Reference electrode was a saturated calomel electrode (SCE) and counter electrode was a platinum foil (10 × 5 mm). The surface topographies of the CS electrodeposited on GCE were recorded using a SUPRA55 field emission scanning electron microscope (FESEM, Germany) Received: July 17, 2015 Accepted: August 29, 2015 Published: August 29, 2015 9481

DOI: 10.1021/acs.analchem.5b02683 Anal. Chem. 2015, 87, 9481−9486

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Analytical Chemistry and a JPK NanoWizard III atomic force microscope (AFM, Germany). The UV spectra of the inclusion complexes, CS-LTrp and CS-D-Trp, were recorded using a UV-2450 UV/vis spectrophotometer (Shimadzu, Japan). The temperature studied in this work for the inclusion complexation of the host−guest system was controlled by an intelligent thermostatic bath (Ningbo Scientz Biotechnology Co., Ltd., China). Electrodeposition of CS or SCS onto GCE. A total of 50 mg of CS was dissolved in 25 mL of 0.1 M HCl, and then a three-electrode system using bare GCE as the working electrode was placed into the 2 mg mL−1 CS solution for 150 s with a constant potential of −0.5 V applied at the working electrode. Electrodeposition of SCS onto GCE was carried out by the same procedure using SCS instead of CS. Here, SCS was synthesized by the method described by Tsai et al.20 Briefly, 2 mL of 1,3-propane sultone was added to 50 mL of 2% acetic acid (w/w) containing 20 mg mL−1 CS under stirring, and then the mixture was allowed to react at 60 °C for 12 h. Next, the mixture was distilled and a large amount of absolute ethanol was poured into the resulting solution for the precipitation of SCS. Finally, the precipitated product was dried in a thermostat oven at 60 °C for 1 day. The process for the synthesis of SCS is shown in Figure 1.

Figure 2. Electrodeposition of CS films on the surface of GCE.

Figure 3. FESEM image of the CS films electrodeposited on GCE.

inclusion complexation with guest molecules. In addition, the AFM images of the electrodeposited CS films are also shown in Figure 4. As can be seen, the CS films exhibit a rough and uneven surface topography with embedded cavities (root-meansquare roughness, 7.385 nm), which can be used for accommodating Trp isomers to be recognized. Electrochemical characterizations of the CS films modified GCE were also carried out in 25 mL of 5 mM Fe(CN)64−/3− by cyclic voltammetry (Figure 5). A pair of well-defined redox peaks attributed to the transition between Fe(CN)64− and Fe(CN)63−, an electroactive probe couple, appears at the CSmodified GCE with a peak-to-peak potential separation (ΔEp) of 106 mV (curve a), while the peak currents at the bare GCE decrease significantly, and the ΔEp is increased to 139 mV (curve b). Generally, the value of ΔEp is related to the electron transfer coefficient,21 and a low ΔEp suggests a fast electron transfer for a single-electron electrochemical reaction.22 The enhanced electron transfer at the CS-modified GCE is attributed to the introduction of CS via electrodeposition. Because 0.1 M HCl was used as the electrolyte for CS electrodeposition, the obtained CS films are positively charged due to protonation. The strong electrostatic interactions between CS and negatively charged Fe(CN)64−/3− result in improved electrochemical behaviors at the CS-modified GCE. The cyclic voltammograms (CV) of the CS-modified GCE also indicate that CS films are formed on the surface of GCE via electrodeposition. Enantiorecognition of Trp Isomers by CS Films Modified GCE. The differential pulse voltammograms (DPV) of L- and D-Trp at the bare GCE are completely overlapped (Figure 6), indicating that the enantiorecognition ability of the bare GCE toward Trp isomers is rather poor. The poor enantioselective ability of the bare GCE can be attributed

Figure 1. Synthetic illustration of SCS.

Electrochemical Enantiorecognition of Trp Isomers. Electrochemical enantiorecognition of Trp isomers with electrodeposited CS and SCS was carried out by differential pulse voltammetry. The as-prepared CS- or SCS-based chiral system was immersed into 25 mL of 0.1 M phosphate buffer solution (PBS) containing 0.5 mM L-Trp or D-Trp (pH 7.0) for 60 s at 25 °C, respectively. After the inclusion complexation was finished, the differential pulse voltammograms (DPV) of these host−guest systems were recorded and compared (CS-LTrp vs CS-D-Trp; SCS-L-Trp vs SCS-D-Trp). Each DPV was repeated five times, and the mean value was calculated for the error bars. To investigate the temperature-sensitive characteristics of the SCS-based chiral system, the host−guest inclusion complexation between SCS and Trp isomers was conducted at different temperatures (0−40 °C).

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RESULTS AND DISCUSSION Electrodeposition of CS Films on GCE. CS is protonated in 0.1 M HCl, so it is positively charged in the solution. When a negative potential of −0.5 V is applied on the GCE, CS is spontaneously deposited onto the surface of GCE due to the strong electrostatic attraction (Figure 2). CS is a kind of natural polysaccharide, and SCS is synthesized by sulfonating CS with 1,3-propane sultone (Figure 1). The CS films electrodeposited on GCE were characterized by FESEM (Figure 3). Obvious and irregular cavities of several hundred nanometers wide are distributed within the adhered CS films, making the CS to be an ideal candidate as host for the 9482

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Figure 4. AFM images of CS films electrodeposited on GCE.

Figure 5. Cyclic voltammograms of 5 mM Fe(CN)64−/3− at CS films modified GCE (a) and bare GCE (b).

Figure 6. Differential pulse voltammograms of 0.5 mM L- and D-Trp at bare GCE in 0.1 M PBS of pH 7.0.

Figure 7. (a) Differential pulse voltammograms of CS-L-Trp and CSD-Trp. (b) Schematic diagram showing the incorporation of L- and DTrp into the double helixes of CS via H-bonds and then penetration through CS films to the electrode surface.

to the fact that GCE functions only as a transducer in the electrochemical measurements, and there are no chiral centers in this system for the enantiorecognition of L- and D-Trp. However, significant differences are observed in both Ep and Ip for CS-D-Trp and CS-L-Trp (Figure 7a), suggesting that efficient recognition can be achieved by the CS films. Compared to CS-D-Trp, CS-L-Trp exhibits a larger Ip, indicating that the amount of L-Trp penetrating through the 2-fold helical structure of CS to the electrode surface is larger than that of DTrp. Also, it is found that the Ep of CS-L-Trp is located negatively compared to CS-D-Trp. The lower Ip and higher Ep of CS-D-Trp imply that the CS preferably includes D-Trp when the hydrophobic indole groups of L- or D-Trp enter the double helixes of CS from the solution. The higher affinity of CS for DTrp can be attributed to the smaller steric hindrance for the

formation of H-bonds between the −OH groups in the double helixes of CS and the −NH2 groups of D-Trp (Figure 7b). Due to the strong inclusion complexation between CS and D-Trp, it is more difficult for D-Trp to penetrate through the CS films to the electrode, resulting in lower Ip and higher Ep compared with that of L-Trp. Theoretical Calculation of Binding Constant. The UV spectra of CS-L-Trp and CS-D-Trp are measured by adding different concentrations of CS (from 0 to 5 mg mL−1) to 0.05 mM (10.2 mg L−1) L-Trp (Figure 8a) and D-Trp (Figure 8b). The absorbance intensity of both CS-L-Trp and CS-D-Trp increases with increasing concentration of CS, suggesting an improved inclusion of L- and D-Trp in the CS via supramolecular interactions.23 The characteristic absorbance of Trp at 278 nm24 is employed to calculate the stoichiometry ratio 9483

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Figure 8. UV spectra of 10.2 mg L−1 L-Trp (a) and D-Trp (b) upon addition of CS of various concentrations at 25 °C, and the CS concentrations from (1) to (11) are 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg mL−1, respectively. Double reciprocal plots of 10.2 mg L−1 L-Trp (c) and DTrp (d) included with CS of various concentrations at 25 °C.

Ep of D-Trp, which implies that the oxidation of D-Trp at the SCS becomes more difficult compared to that at CS. As discussed by Tsai et al.,20 the intramolecular H-bonds of CS existing in the form of O···H···O are destroyed after the sulfonation treatment, and the number of free −OH groups within the double helixes of SCS is increased, which can bind a larger amount of D-Trp via the intermolecular H-bonds of O··· H···N and result in less D-Trp penetrating through the SCS films to the electrode. However, sulfonation of CS films has little influence on the inclusion complexation between SCS and L-Trp due to the significant steric hindrance generating in the formation of intermolecular H-bonds in the host−guest system (Figure 9b). As a result, the enantiorecognition efficiency at the SCS-modified GCE is remarkably enhanced compared with that at the CS-modified GCE. The chiral recognition process within the SCS can be simply illustrated as Figure 9c. Temperature Sensitivity of the SCS-Based Chiral System. Both intramolecular H-bonds and intermolecular Hbonds are influenced significantly by temperature, so the chiral recognition of Trp isomers by SCS may be temperaturedependent. In fact, temperature-sensitive enantiorecognition of Trp isomers based on another polysaccharide, β-cyclodextrin4 or modified β-cyclodextrin,5 has been reported by our group. The influence of temperature on the recognition efficiency (differences in Ep and Ip) is also studied in this work (Figure 10). It seems that the highest recognition efficiency is obtained at 25 °C. Although most intramolecular H-bonds are destroyed during the sulfonation of CS, there still exist intramolecular Hbonds within the double helixes of SCS,7 which are stable enough at low temperatures (below 25 °C), resulting in difficult host−guest inclusion complexation and low recognition efficiency. However, an excessively high temperature (from 30 to 40 °C) will lead to the breakage of the intermolecular H-

and binding constant of CS-L-Trp and CS-D-Trp according to the following equation proposed by Benesi and Hildebrand:25 1 1 1 = + A − A0 Δε × [Trp] (Δε × [Trp] × K × [CS]n0 )

where A is the absorbance of L- or D-Trp at each CS concentration and A0 represents the absorbance in the absence of CS. Δε is the differential molar extinction coefficient of L- or D-Trp in the absence and presence of CS. K is the binding constant between CS and Trp isomers. [CS]n0 and [Trp] are the original concentrations of CS (from 0 to 5.0 mg mL−1) and Trp isomers (10.2 mg L−1), respectively, and n represents the stoichiometry ratio of the inclusion complex. Different values of n were tested to give the plots of 1/ΔA versus 1/[CS]n0, and a straight line is obtained only when the value of n is equal to 1 (Figure 8c,d), indicating formation of the 1:1 complex between CS and L- or D-Trp. The value of K was calculated to be 0.059 (CS-L-Trp) and 0.081 (CS-D-Trp), respectively, by dividing the intercept by the slope of the plots in Figure 8c,d. The larger value of K of CS-D-Trp indicates that CS exhibits higher affinity to D-Trp than L-Trp, which is attributed to the fact that, compared with L-Trp, more stable intermolecular H-bonds are formed between CS and D-Trp due to the smaller steric hindrance. Enantiorecognition of Trp Isomers by Sulfonated CS (SCS). To obtain further information regarding the host−guest interactions, SCS was used as the host instead of CS. It is interesting to find that, compared with CS, the recognition efficiency at SCS is significantly improved (Figure 9a). By comparing Figures 7a and 9a, it is observed that, for L-Trp, both Ep and Ip remain almost unchanged at CS- and SCS-modified GCE, and the improvement in the recognition efficiency is surely caused by the decrease in Ip as well as the positive shift of 9484

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Figure 9. (a) Differential pulse voltammograms of SCS-L-Trp and SCS-D-Trp. (b) Illustration showing the breakage of intramolecular H-bonds in the double helixes of CS via sulfonation and the formation of intermolecular H-bonds between SCS and Trp isomers. (c) Simple illustration showing the chiral recognition process within SCS.

bonds between SCS and Trp isomers formed during the inclusion, which in turn deteriorates the enantiorecognition efficiency toward Trp isomers. Chiral Recognition of Trp Isomers in Racemic Solution. Whether or not a chiral system exhibits the ability of predicting the ratio of L- and D-isomers in racemic mixture is of great importance for its further practical applications,3 however, most enantiorecognition was carried out in separated isomer solution. Here, we tried to resolve the mixture of L- and D-Trp with a total concentration of 0.5 mM by the SCSmodified GCE. It is amazing that the two oxidation peaks of Land D-Trp merge into a broad one, regardless of the relative content of D-Trp (D-Trp%) in the mixture. Fortunately, it is found that the Ep of the broad oxidation peak shifts positively with increasing D-Trp%, and there is an obvious linear relationship between Ep and D-Trp% in the mixture (Figure

Figure 10. Influence of temperature on the enantiorecognition efficiency of SCS toward Trp isomers. Error bars represent standard deviation for five independent measurements.

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ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of China (21275023, 11532003), Natural Science Foundation of Jiangsu Province (BK2012593), Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology (BM2012110), Graduate Research Innovation Program for Universities of Jiangsu Province (SJLX15_0535), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

11). This result implies that it is possible to accurately predict D-Trp% in the mixture of Trp isomers by the proposed SCS-

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based chiral system. The theoretical and determined compositions (L-/D-Trp) by the SCS-modified GCE are listed in Table 1, and the rather small standard deviations (SDs) indicate that the proposed chiral sensor is reliable. Table 1. Theoretical and Determined Compositions (L-/DTrp) and Standard Deviations (SD) by the SCS-Modified GCE determined composition

SD (%) (n = 5)

90:10 70:30 60:40 50:50 40:60 30:70 20:80 10:90

90.13:9.87 71.72:28.28 61.19:38.81 50.56:49.34 42.77:57.23 32.24:67.76 23.03:76.97 9.88:90.12

0.32 0.45 0.31 0.38 0.36 0.39 0.29 0.36

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CONCLUSIONS To summarize, we have designed a novel chiral system based on the 2-fold helical conformation of CS for selectively recognized D-Trp. The DNA-inspired enantiorecognition is achieved via selective formation of intermolecular H-bonds due to the different steric hindrance in the host−guest system, which is further verified by the recognition of Trp isomers with SCS. In the double helixes of SCS, most intramolecular Hbonds are destroyed by sulfonation, resulting in strengthened intermolecular H-bonds between SCS and D-Trp and enhanced recognition efficiency. The temperature-sensitive property of the chiral system is also reported. More importantly, the SCSbased chiral system exhibits the ability of predicting the ratio of L- and D-Trp in a racemic mixture, showing its potential in future practical applications in fields such as drug detection and analysis.26,27

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REFERENCES

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Figure 11. Relationship between Ep (black) and Ip (red) and different D-Trp% in the mixture of Trp isomers. Error bars represent standard deviation for five independent measurements.

theoretical composition

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*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. 9486

DOI: 10.1021/acs.analchem.5b02683 Anal. Chem. 2015, 87, 9481−9486