Dual-Signal Electrochemical Enantiospecific Recognition System via

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Dual-Signal Electrochemical Enantiospecific Recognition System via Competitive Supramolecular HostGuest Interactions: The Case of Phenylalanine Yinhui Yi, Depeng Zhang, Yuzhi Ma, Xiangyang Wu, and Gangbing Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05047 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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Analytical Chemistry

Dual-Signal Electrochemical Enantiospecific Recognition System via Competitive Supramolecular Host-Guest Interactions: The Case of Phenylalanine Yinhui Yi,a Depeng Zhang,a Yuzhi Ma,a Xiangyang Wu,a Gangbing Zhua,b,c a

School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, P. R.

China b

State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha

410082, People’s Republic of China c

Department of Applied Biology and Chemical Technology, and the State Key Laboratory of

Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

ABSTRACT For developing highly selective and sensitive electrochemical sensors for chiral recognition, taking advantage of the synthetical properties of β-cyclodextrin (β-CD, strong host-guest recognition) and carbon nanotubes wrapped with reduced graphene oxide (CNTs@rGO, excellent electrochemical property and large surface area) as well as the differences in binding affinity between β-CD and guest molecules, a dual signal electrochemical sensing strategy was proposed herein for the first time in chiral recognition based on the competitive host-guest interaction between probe and chiral isomers with β-CD/CNTs@rGO. As a model system, rhodamine B (RhB) and phenylalanine enantiomers (D-

 Corresponding author. Tel.: (+86)-511- 88790955; Fax: (+86)-511- 88790955.

E-mail address: [email protected] 1 / 33

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and L-Phe) were introduced as probe and target enantiomers respectively. Due to the host-guest interaction, RhB can enter into the β-CD cavity, showing remarkable oxidation peak current of RhB. In the presence of L-Phe, competitive interaction with the β-CD cavity occurs and RhB are replaced by L-Phe owing to the stronger binding affinity between L-Phe and β-CD, which resulted that the peak current of RhB decreases and peak current of L-Phe appears, and interestingly, the changes of both signals linearly correlate with the concentration of L-Phe. As for D-Phe, it cannot replace RhB owing to the weaker binding affinity between D-Phe and β-CD. Based on this, a dual-signal electrochemical sensor was developed successfully for recognizing Phe. This dual-signal sensing strategy can provide highly selective and sensitive recognition compared to single-signal sensor and has important potential applications in chiral recognition. KEYWORDS: Chiral recognition; enantiomers; cyclodextrin; phenylalanine; electrochemical sensors; graphene.

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INTRODUCTION Chirality has an important impact on chemical/biological research, since most active substances possess chirality. Generally, the performance of chiral enantiomers show great differences in terms of biochemical activity, toxicity, transport processes and metabolic pathways.1-3 Typically, only one isomer

exhibits perfect activity and the other has no desirable value and even causes serious side-

effect. Thus, chiral recognition is always a popular topic in chemical and biological research.4-6 For resolving this problem, many approaches, such as capillary electrophoresis,7 high-performance liquid chromatography,8 circular dichroism spectroscopy,9, 10 colorimetric11, 12 and fluorescent13, 14, were developed to recognize electroactive chiral molecules, which are very important in chiral recognition. However, the most of reported approaches need complex sample pretreatment and expensive chiral columns as well as time-consuming.15, 16 Recently, electrochemical chiral recognition has received considerable attention,

due to the

many advantages they offer , such as low cost, fast response, inexpensive instrument, and facile miniaturization.17-22 For instance, Kong et al.23 demonstrated that the self-assemblies composed of diphenylalanine and oxalic acid with different charging states possess quite different chiral recognition capabilities towards tryptophan isomers. In another interesting report, the researchers constructed an electrochemical chiral sensing platform for D-/L-tryptophan, D-/L-3,4dihydroxyphenylalanine and (R)-/(S)-propranolol by electrodepositing R/S-2’-hydroxymethyl-3,4ethylenedioxythiophene on electrode surface.16 However, almost all of these methods can only present the ability to measure the percentage of chiral enantiomers in the racemic mixture, which cannot directly achieve the selective and quantitative determination.15,

16, 24

Furthermore, these

electrochemical sensors usually involves only one signaling mechanism, which may not be reliable enough since some factors (such as detection surroundings and mistakes) can interfere with the 3 / 33



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detection results, while sensing platform with multiple response signals can make the detective results more convincing, and it is expected to provide additional responsive information and result in the improvement of the selectivity and sensitivity of the sensors.25-27 Therefore, developing an effective approach with multiple signals to achieve selective and sensitive quantitative determination for chiral enantiomers is of great significance and has important potential applications. Cyclodextrins (CDs) are a class of cyclic oligosaccharides consisting of 6, 7 or 8 glucose units (named α-, β- or γ-CD, respectively), which are toroidal in shape with a hydrophobic inner cavity and a hydrophilic exterior.28-30 Generally, CDs present a relatively low cost and an excellent capability to host effectively, selectively and enantio-selectively various compounds into their hydrophobic cavities to form host-guest inclusion complexes.28, 31, 32 These interesting characteristics make them have important applications in constructing chiral sensors, and it is true that several CDs-based electrochemical methods have been developed for recognizing electroactive chiral molecules.33, 34 As described in the second paragraph, almost all of the developed electrochemical proposals including CDs-based sensors for chiral recognition cannot achieve directly the selective and quantitative determination, and they usually involved in only one signaling mechanism. In fact, CDs have been widely applied in the field of electroanalytical chemistry presently, while the reported works based on CDs were usually focused on single signal sensing.35-37 Recently, it has been reported that there are some electroactive molecules, whose binding affinity with CDs cavities are in-between different enantiomers with CDs,38-40 this insight makes us convinced that CDs can be used to fabricate multiple signaling sensor for achieving quantitative determination of chiral enantiomers. However, only using pure CDs to construct directly electrochemical sensors is not desirable. The requirement for an effective electrochemical chiral sensor is not only discrimination of each enantiomer but also one material distinguishing and improving the responsive signal. The alliance of 4 / 33


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enantioselectivity with electrochemical properties endows the conjugated materials with many interesting characteristics in serving as sensing devices.

32, 41, 42

One single-atom thick two-

dimensional nanostructure, graphene, which has attracted considerable attention in many fields due to its high conductivity, large surface area, good biocompatibility and low cost. However, pure graphene tends to form agglomerates and even restack to shape graphite, which can largely lessen its effective surface area and electrochemically active sites.43-46 Recent reports demonstrated that coreshell heterostructure carbon nanotubes wrapped with reduced graphene oxide (CNTs@rGO) nanostructure can show the synergetic performance of rGO and CNTs: the outer rGO shell can increase the contacting area and provide lots of area-normalized edge-plane structures as well as active sites; the CNTs core can impede effectively the rGO aggregation and facilitate the electron transfer rate inside the nanocomposites.47,

48

Consequently, CNTs@rGO may be a desirable

nanomaterial to ally CDs for constructing electrochemical chiral sensors. It is reported that different forms of phenylalanine (Phe) enantiomers have different effects in humans, thus the effective recognition of D- and L-Phe is highly crucial.49-52 Herein, by choosing electroactive substance Rhodamine B (RhB) and Phe enantiomers as model probe and analyte, respectively, a novel electrochemical sensing platform with dual-signals was developed for the selective and selective quantitative determination of chiral molecules based on a competitive hostguest interaction between different guest molecules with β-CD (Scheme 1), which

comprises of

four steps as follows: ❶ producing CNTs@rGO nanostructure (Scheme S1), ❷ electro-oxidizing β-CD on CNTs@rGO electrode surface, ❸ host-gust interaction between β-CD with RhB, and ❹ competitively replacing probe (RhB) by Phe. The stronger binding affinity of L-Phe with β-CD makes L-Phe replace RhB in β-CD cavities, but D-Phe can’t replace RhB since its binding affinity with βCD is weak. The research results demonstrated that when the |ΔIRhB|+|ΔIL-Phe| (ΔI, the change values 5 / 33



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of the peak current) value is adopted as the response signal, the proposed dual signal strategy has more superior detection limit, sensitivity and linear range than single signal strategy (|ΔIL-Phe| as signal), and it can be used for the directly selective and sensitive quantitative determination of enantiomers. Besides, this work promotes the CDs-based electrochemical sensors from single signal to dual signals, which is of great significance in accelerating the development of CDs-based electroanalytical chemistry.

EXPERIMENTAL SECTION Reagents and Materials Phe enantiomers and β-CD were purchased from Alfa Aesar (USA) and Beijing Chemical Reagent factory, respectively. N-acetylaniline (NAANI) was obtained from Shanghai Chemical Reagents and used upon recrystallization in ethanol. CNTs were purchased from Shenzhen Nanotech Port Ltd, China. 0.1 M phosphate buffer solution (PBS) was used as supporting electrolyte. The other chemical reagents are of analytical grade and directly used without further purification. Apparatus The electrochemical test was performed on a CHI 660E Electrochemical Workstation (Chenhua Instrument Company of Shanghai, China). A 3-electrode cell was used with a platinum wire (counter electrode), Ag/AgCl electrode (reference electrode) and glass carbon electrode (GCE, working electrode). The morphology and structure of the produced nanomaterials were characterized by transmission electron microscopy (TEM, JEM-3010, Joel, Japan) and scanning electron microscopy (SEM, JSM-6360LV, USA), Fourier transform infrared spectroscopy (FT-IR, NICOLET 6700, Thermo Fisher Scientific Inc., USA) and thermogravimetric analysis (TGA, NETZSCH STA 409 PC) Unless otherwise stated, the electrochemical test toward Phe enantiomers was carried out in 0.1 6 / 33


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M PBS (pH 7.0) at room temperature (298±2 K), and the electrochemical impedance analysis (EIS) of modified electrodes was carried out in KCl aqueous solution containing Fe[CN]63-/4- as a redox marker. Construction of Electrochemical Chiral Sensing Platform for Phe Enantiomers CNTs@rGO: before preparing CNTs@rGO, the CNTs@GO (carbon nanotubes-graphene oxide) nanohybrids were synthesized via longitudinal partially unzipping CNTs according to the previous report.48 In brief, 100.0 mg CNTs was dispersed in 24.0 mL concentrated sulfuric acid (98 wt%) to be stirred for 1 h at room temperature. Then, 2.5 mL H3PO4 (85 wt%) was added and the mixture were continuously stirred 18 min before adding 300.0 mg KMnO4. Next, the mixture solution was heated for 2 h at 65 °C, and then poured into150.0 mL ice water containing 4.0 mL H2O2 (30 % vol). The final solution was filtered, washed and dried in vacuum oven over night. For preparing CNTs@rGO nanohybrids, 300.0 μL NH3·H2O and 20.0 μL N2H4 solution were slowly added to a 20.0 mL CNT@GO dispersion (0.5 mg mL−1) under stirring, and then, this solution was mixed via sonication followed by heating under magnetic stirring at 60 °C for 24 h. Finally, the formed stable black dispersion was filtered, washed, and then dried to obtain CNTs@rGO. β-CD/CNTs@rGO Modified Electrode: GCE was polished carefully with alumina powder to a mirror-like plane and rinsed with ultrapure water, followed by ultrasonication in acetone and ultrapure water, then GCE was dried under N2 atmosphere. 10.0 μL CNTs@rGO suspension (0.5 mg mL-1) was dropped on GCE surface and dried with infrared lamp. Next, CNTs@rGO/GCE was kept at fixed-potential of 1.0 V for 2 min in 0.1 M NAANI+1.0 M HClO4 solution, and then it was scanned from -0.2 to 1.0 V for 25 cycles. Finally, by electrically oxidizing the electrode at 1.2 V for 18 min in DMSO containing 0.05 M β-CD and 0.1 M LiClO4, β-CD/CNTs@rGO modified electrode (βCD/CNTs@rGO/GCE) was prepared. In this work, the other modified electrodes were obtained with 7 / 33



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similar procedures for comparison. Electrochemical

Test

for

Phe

Enantiomers:

Before

testing

electrochemically,

β-

CD/CNTs@rGO/GCE was incubated in 0.1 mM RhB solution for a definite time to make RhB molecules enter fully into β-CD cavities, and gently rinsed with ultrapure water; the produced electrode

was

denoted

as

RhB-bound

β-CD/CNTs@rGO/GCE.

Then,

RhB-bound

β-

CD/CNTs@rGO/GCE was incubated further in 0.1 M PBS containing D- or L-Phe with different concentrations, and rinsed gently with ultrapure water. After that, the electrochemical response was measured by differential pulse voltammetry (DPV) in 0.1 M PBS.

RESULTS AND DISCUSSION Characterization of Various Nanohybrids and Modified Electrodes Raman spectroscopy (Figure S1) and TGA (Figure S2) were firstly used to study CNTs@rGO. Then, TEM and SEM were performed to reveal the detailed morphology and structure of CNTs@rGO and corresponding modified electrodes. Figure S3A shows representative TEM image from CNTs. After unzipping CNTs, the morphology obviously changed with an increased width, and the formed CNTs@rGO exhibits core-shell nanostructure coupled with the core of CNTs and the shell of graphene. Figure 1 shows the SEM images of CNTs, CNTs@rGO, β-CD and β-CD/CNTs@rGO modified GCE, it is noted that the results from image A and B are consistent with those from Figure S1. As for β-CD/GCE (Figure 1C), a very uniform, smooth and dense β-CD film was formed on electrode surface. While for the β-CD/CNTs@rGO modified electrode (Figure 1C), a rough surface feature with nanometer scale bulges was observed. The β-CD/CNTs@rGO film was further investigated by Fourier transform infrared spectroscopy (FT-IR). As shown in Figure S4, CNTs shows representative carbon-carbon single band and carbon-carbon double bond at 1070 and 1632 8 / 33


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cm−1, respectively. After transformation to CNTs@GO, the produced nanohybrids exhibit the characteristic peaks of oxygen groups including epoxide, hydroxy and carboxy groups at 813, 1400 and 1715 cm−1. Curve c shows the FT-IR spectra of CNTs@rGO, it is noted that the characteristic peaks from the oxygen groups are almost disappearing owing to the successful reduction of GO via NH3·H2O and N2H4. Compared with CNTs@rGO, the FT-IR spectrum of β-CD/CNTs@rGO shows new and typical β-CD absorption features of CH2 stretching vibrations at 2925 cm-1 and the coupled C-O/C-C stretching/O-H bending vibrations at 1020 and 1078 cm-1. These results suggested that CNTs@rGO nanohybrids were successfully prepared via unzipping CNTs and modified with β-CD through the electro-oxidation procedure. EIS has gained widespread application in characterizing functionalized electrode surfaces and the transduction of sensors.53 For demonstrating the conductivity of the modified electrodes, the electron transfer behavior of β-CD/CNTs@rGO/GCE was investigated by EIS. Figure 2A exhibits the EIS results of β-CD/GCE and β-CD/CNTs@rGO/GCE recorded in 0.1 M KCl aqueous solution containing 5.0 mM Fe[CN]63-/4-. It is found that the fitted charge transfer resistance (Rct) value for βCD/GCE (~900 Ω) is much larger than that of the bare GCE (~263 Ω) and CNTs@rGO/GCE (its Rct value is too small to almost be ignored) owing to the poor conductivity of β-CD. However, for βCD/CNTs@rGO/GCE, its Rct value is reduced dramatically, demonstrating that CNTs@rGO can exhibit excellent electrochemical properties to improve the conductivity of the modified electrode and promote the electron transfer. Cyclic voltammetry (CV, Figure 2B) analysis was further used to substantiate the results obtained in Figure 2A. It is noted from Figure 2B(a) that a well-defined redox couple appeared at GCE with the peak separation of ~106 mV. After GCE modification with CNTs@rGO, the redox peak current was significantly improved with a ~85 mV peak separation value owing to excellent conductivity, large specific surface area and electrochemical reversibility of 9 / 33



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CNTs@rGO. As for β-CD/GCE, a weak redox couple with a large peak separation (~250 mV) value was observed. Figure 2B(d) shows the CV response of β-CD/CNTs@rGO/GCE, an obvious redox peak with a small peak separation was obtained owing to the presence of CNTs@rGO. These results are in agreement with those obtained by EIS analysis. Electrochemical Enantioselective Recognition of Phe Enantiomers The feasibility of the proposed sensor herein for electrochemically recognizing Phe enantiomers was demonstrated in detail. Firstly, the electrochemical responses of D- and L-Phe at various modified electrodes were studied via DPV. As shown in Figure 3, it can be found from Figure 3A that both Dand L-Phe exhibit a weak and similar oxidation peak on bare GCE at ~0.674 V resulting from their similar structures and chemical properties. Figure 3B shows the DPV responses of Phe enantiomers at β-CD/GCE, the peak current from D- and L-Phe increased with different degree owing to the hostguest recognition capability of β-CD and different binding affinities of β-CD with D-/L-Phe, while there is no peak potential difference observed. CNTs@rGO introduced in this work was designed to improve the sensing performance of the developed proposal. Figure 3C shows the DPV responses of Phe enantiomers at β-CD/CNTs@rGO modified electrode, it is noted that the peak current of D- and L-Phe increased further and the peak potential shifted negatively to ~0.636 V compared to those at β-CD/GCE owing to the excellent conductivity and large surface area of CNTs, but the peak potential difference was still not presented. Furthermore, the electrochemical response of Phe enantiomers at CNTs@rGO/GCE was also investigated (Figure 3D). The similar and large peak currents of D- and L-Phe were observed. These phenomena demonstrate that β-CD/CNTs@rGO/GCE can show the synergetic advantages of β-CD and CNTs@rGO in electrochemically sensing Phe enantiomers, but it cannot be used to achieve the direct quantitative and selective recognition of Phe enantiomers. Next, the DPV response of β-CD/CNTs@rGO/GCE after being separately incubated in various 10 / 33


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analytes were measured in 0.1 M PBS (Figure 4). Figure 4 reveals that single RhB and Phe enantiomer shows oxidation peak at ~0.898 V and 0.636 V, respectively. However, in the presence of L-Phe, it is found from Figure 4A that the peak current of RhB at β-CD/CNTs@rGO/GCE decreases remarkably and meanwhile an obvious peak current of L-Phe appears owing to the competitive association of L-Phe/RhB to β-CD and the replacement of RhB probe by L-Phe (curve c). This indicates that RhB-bound β-CD/CNTs@rGO/GCE can be used to construct dual-signaling electrochemical sensor for L-Phe. Figure 4B shows that the peak current of RhB at βCD/CNTs@rGO/GCE in the presence of D-Phe (curve c) is almost as high as that shown in the curve a, suggesting that D-Phe cannot displace the RhB molecules in β-CD cavities. In addition, the DPV responses of RhB without (a) and with L-Phe (b) solution at β-CD/GCE were studied for comparison (Figure S5), it was noted that the corresponding peak current are obviously weaker than those at βCD/CNTs@rGO/GCE. Based on the above experiments, we can conclude that RhB bound βCD/CNTs@rGO/GCE can be used to fabricate directly quantified and selective dual signal sensor for Phe enantiomers (L-Phe). The mechanisms of the proposed dual signal electrochemical sensor for the chiral recognition of Phe isomers were summarized as follows: Owing to the host-guest interaction, RhB probe can enter into the β-CD inner cavity and the RhBbound β-CD/CNTs@rGO modified electrode presents a remarkable oxidation peak current of RhB. Due to the stronger binding affinity between L-Phe and β-CD, the competitive association to β-CD cavities happens and the RhB would be replaced by L-Phe in the presence of L-Phe, which resulted in the decrease of the peak current of RhB and appearance of the peak current of L-Phe. Thus, the electrochemical sensing platform with dual-signals for L-Phe can be constructed based on the change values of the peak current from both RhB and L-Phe via the concentration of L-Phe. Since the binding 11 / 33



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affinity between D-Phe and β-CD is weak, D-Phe cannot replace the RhB probe. Based on the principle above, the dual signal electrochemical sensing platform for recognizing Phe isomers was developed by using RhB-bound β-CD/CNTs@rGO electrode. Optimization of The Conditions for Chiral Recognition In order to achieve the maximum efficacy of the proposed sensor for recognizing L-Phe, some control experiments including the electro-oxidation time of β-CD and the incubation time of modified electrodes in RhB/L-Phe solutions were carried out to optimize the detection conditions. The amount of β-CD on electrode surface is very important to improve the sensitivity and chiral recognition capability for the sensor, thus the electro-oxidation time of β-CD needs to be optimized. Figure 5A shows the effect of the electro-oxidation time of β-CD on the DPV peak current of βCD/CNTs@rGO/GCE after incubation in L-Phe or RhB solutions. Owing to the host-guest recognition interaction between β-CD and L-Phe (or RhB), it was noted that the DPV peak current of RhB (curve a) and L-Phe (curve b) increased with the increase of the electro-oxidation time, and the best value was presented at 18 min, hence, this time was used as the optimum electro-oxidation time for β-CD. In addition, the incubation time of β-CD/CNTs@rGO/GCE in RhB and L-Phe solution is a key parameter that will increase the sensor sensitivity and save operation time as well as affecting the sensing performance of the electrode. Figure 5B shows that the oxidation peak currents of the modified electrodes toward RhB (0.1 mM) and L-Phe (4.0 μM) increased with increase in incubation time, and they reached maximum values at 110 s for RhB and 210 s for L-Phe respectively. Therefore, 110 s and 210 s are selected as the optimal time for incubating the obtained electrode in RhB and LPhe solution, respectively. Quantification Recognition of L-Phe 12 / 33


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Under the optimized conditions, the quantitative determination of L-Phe based on RhB-bound βCD/CNTs@rGO/GCE was carried out by DPV and the corresponding results were shown in Figure 6. It is seen that the RhB probe in β-CD cavities is replaced gradually by L-Phe with the increase of L-Phe, which makes the oxidation peak current of RhB decrease gradually from curve a to h in the range of L-Phe concentration from 50.0 nM to 22.0 μM. For the peak current from L-Phe, it increases gradually from the curve b to h, but no obvious oxidation peak current of L-Phe can be observed from the curve b, which is probably due to the small concentration of L-Phe. By combining the current decrease of RhB and increase of L-Phe as dual-signal (|ΔIRhB|+|ΔIL-Phe|) to detect quantitatively the concentration of L-Phe, the results show that the |ΔIRhB|+|ΔIL-Phe| value linearly increases with the increase of L-Phe concentration from 50.0 nM to 22.0 μM, and the linear regression equation is |ΔIRhB|+|ΔIL-Phe| (μA)=2.694+1.106C (μM) (R2=0.992) with a LOD (limit of detection) of 0.013 μM (S/N=3, 95% confidence interval) (Figure 6B). For comparison, the linear range on the basic singlesignal solely caused by the peak current change of L-Phe was calculated (Figure 6C). The linear range was 0.2-13.0 μM and the corresponding linear regression equation was (|ΔIL-Phe| (μA) = 0.582 + 0.425 CL-Phe (μM) (R2=0.997) with a LOD of 0.08 μM, which is narrower relatively than that obtained via a dual signal strategy. It is obvious that the linear range and LOD value obtained through dual signal strategy are much better than those obtained through single-signal method. Chiral Recognition of Phe Isomers in Racemic Mixture The recognition applicability of the proposed sensor was further studied by detecting the concentration of L-Phe in racemic mixture composed of D- and L-Phe. Specifically, the quantitive determination of L-Phe based on RhB-bound β-CD/CNTs@rGO/GCE was carried out in the presence of D-Phe. Definite amounts of L-Phe were spiked in PBS containing 5.0 μM D-Phe, and the concentrations of L-Phe in each sample were measured by the proposed method (Table 1). It was 13 / 33



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found that the recovery for L-Phe is in the range of 95.0-104.7%, indicating that the developed sensor strategy is satisfactory to achieve the quantitative recognition for Phe enantiomers. Obviously, compared to the previous reports for chiral recognizing the percentage of D-/L-Phe isomers in racemic mixture coupled with single signal, the as-developed dual signal sensor in this work can achieve directly the qualitative and quantitative determination for L-Phe with high sensitivity. Reproducibility and Stability The reproducibility of the developed dual-signaling sensor was evaluated by detecting the DPV peak current response to six samples containing 4.0 μM L-Phe (Figure S6A). The results show that the sensor has a superior reproducibility coupled with a relative standard deviation of 6.27%. The stability of the dual-signaling sensor was studied after storing the sensor at 4 °C for 1 week, the result show that a small peak current decrease (about 7.43%) was noted; after storing 5 weeks, 86.2% of the initial current response was still remained (Figure S6B). These results demonstrate that the proposed dual signaling sensor for chiral recognition has satisfactory reproducibility and stability.

CONCLUSIONS In this work, by electro-oxidizing β-CD on CNTs@rGO electrode surface and using RhB as probe, a novel dual-signal electrochemical sensing strategy was developed for the first time in chiral recognition based on the synergetic properties of CNTs@rGO and β-CD as well as different bonding affinity between β-CD with different targets. Choosing Phe enantiomers as model analytes, the experimental results demonstrated that the proposed sensor can achieve selective and quantified determination of chiral enantiomers in racemic mixture, and the dual-signaling sensor has wider linear range and lower LOD than single-signaling sensor. In fact, the as-proposed novel sensor, based on the principle of competitive host-guest interaction between probe and enantiomers with β-CD, can 14 / 33


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also be used to recognize non-electroactive chiral enantiomers (such as D-/L-tetramisol and many non-electroactive chiral pesticides) which are reported in few corresponding papers up to now. It is believed that this work has important promising applications for recognizing Phe enantiomers; meanwhile, this work is of great significance for developing new and effective methods for chiral recognition and accelerating the development of CD-based electroanalytical chemistry from single signal to multiple signals. ACKNOWLEDGMENTS We acknowledge the support from the National Natural Science Foundation of China (21607061), the Opening Project of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (2016017), the Priority Academic Program Development of Jiangsu Higher Education Institutions, Collaborative Innovation Center of Technology and Material of Water Treatment. And we also thank the reviewers for their valuable and insightful comments. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXXX The schematic procedures for preparing CNTs@rGO; characterization (Raman spectra, TGA, TEM and FT-IR spectra) of various nanohybrids; DPV responses of RhB and L-Phe+RhB at β-CD/GCE; table for the sensor’ reproducibility and stability. (PDF)

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Figure captions Scheme 1. Schematic illustration of the dual-signaling electrochemical chiral recognition for Phe enantiomers based on the competitive host-guest interaction between β-CD cavities and RhB (probe)/Phe (target). Step ❶ for preparing CNTs@rGO was shown in Scheme S1. Figure 1. SEM images of (A) CNTs, (B) CNTs@rGO, (C) β-CD/GCE and (D) βCD/CNTs@rGO/GCE. Figure 2. (A) EIS plots and (B) cyclic voltammograms of bare GCE (a), CNTs@rGO/GCE (b), βCD/GCE (c) and β-CD/CNTs@rGO/GCE (d) recorded in Fe[CN]63-/4- aqueous solution. The impedance spectra were recorded in the range of 100 kHz-0.1 Hz at a potential of 0.24 V and AC amplitude of 5.0 mV. Figure 3. DPV responses of (A) the bare GCE, (B) β-CD/GCE, (C) β-CD/CNTs@rGO/GCE, and (D) CNTs@rGO/GCE in the presence of D- (curve a) and L-Phe (curve b) in 0.1 M PBS. Figure 4. DPV responses of (A) RhB (a), L-Phe (b), L-Phe+RhB (c), and (B) RhB (a), D-Phe (b), DPhe+RhB (c) solution at β-CD/CNTs@rGO/GCE. Figure 5. (A) Effect of the electro-oxidation time of β-CD on the DPV peak current of βCD/CNTs@rGO/GCE in 0.1 M PBS after incubation in 4.0 μM (a) RhB and (b) L-Phe solutions. (B) Effects of incubation time on the DPV peak currents of β-CD/CNTs@rGO/GCE in PBS after incubation in (a) 0.1 mM RhB and (b) 4.0 μM L-Phe solutions. Figure 6. (A) DPV responses recorded at RhB-bound β-CD/CNTs@rGO/GCE E in 0.1 M PBS upon incubation with different concentrations of L-Phe (from a to h): 0, 0.05, 0.2, 1.5, 5.0, 10.0, 15.0, and 22.0 μM. (B) The calibration plots of the concentration of L-Phe vs. dual-signal change (|ΔIRhB|+|ΔILPhe|).

(C) The calibration plots of the concentration of L-Phe vs. single signal change from L-Phe.

Table 1. The chiral recognition of L-Phe in the racemic mixture composed of D-Phe and different 23 / 33



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concentrations of L-Phe.

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Scheme 1.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Table 1. Racemic mixture

Added [M]

Found [M]

Recovery [%]

a

1.0

0.96

96.0

b

5.0

4.75

95.0

c

10.00

10.47

104.7

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Page 33 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only

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Dual-Signal Electrochemical Enantiospecific Recognition System via

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