Electroactive Au@Ag NP Assemblies Driven Signal Amplification for

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Electroactive Au@Ag NP Assemblies Driven Signal Amplification for Ultrasensitive Chiral Recognition of D-/L-Trp Yuan Zhao, Linyan Cui, Wei Ke, Fangjie Zheng, and Xiu Li ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06040 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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ACS Sustainable Chemistry & Engineering

Electroactive Au@Ag NP Assemblies Driven Signal Amplification for Ultrasensitive Chiral Recognition of D-/L-Trp Yuan Zhao,*, †,‡ Linyan Cui,†,‡ Wei Ke,†,‡ Fangjie Zheng,†,‡ Xiu Li,§ †Key

Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical

and Material Engineering, Jiangnan University, Wuxi, Jiangsu 214122, China. ‡International

Joint Research Center for Photoresponsive Molecules and Materials, School of

Chemical and Material Engineering, Jiangnan University, Wuxi, 214122, China. §School

of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.

Corresponding author. E-mail: [email protected]

ABSTRACT: A novel ingenious and ultrasensitive chiral electrochemical transducer is proposed for the tryptophan (Trp) isomers detection by using electroactive Au@Ag NPs as electrochemical tags. Moreover, the large binding constant of D-Trp on NPs and strong interaction between D-Trp and Cu2+ cause electroactive Au@Ag NP to assembly on the electrode, generating strong differential pulse voltammetry (DPV) signals from the oxidation of Ag0 to Ag+. In sharp contrast to D-Trp, L-Trp leads to the assembly of Au@Ag NP oligomers on electrode, resulting in a weak DPV signal. The distinct DPV responses enable the developed electrochemical chiral transducer for the sensitive and accurate quantification of D-/L-Trp. The limit of detection (LOD) is 1.21 pM for D-Trp. This established electrochemical chiral sensor also achieves the specific determination of enantiomeric excess. In comparison to other reported approaches, this proposed electrochemical chiral sensor excels by its sensitivity, simplicity and good availability of electroactive Au@Ag NP assemblies. Target-induced colorimetric assays can be converted into electrochemical assays for the dual signal amplification in the field of ultrasensitive enantioselective chiral discrimination. KEYWORDS: Electroactive, Au@Ag NPs, Assemblies, Chiral recognition, Trp enantiomers

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INTRODUCTION In nature, chiral amino acids (AAs) are important in the metabolism.1 2One of amino acid isomers is an essential material for human life, however the others often exhibit a different biochemical effect and induce serious side-effect.3-5 Generally, only L-AAs are used to express to functional and structural proteins, but D-AAs are relevant to some diseases.6,7

Thus, the recognition of

D-AAs from L-AAs is necessary in the field of biology and chemistry. As we all known, Trp is regarded as precursor of neurotransmitter serotonin , therefore, the content of L-Trp has a close relationship with the hepatic disease.8 The preparation of peptide antibiotics and immunosuppressants agents needs D-Trp as intermediates.9 Because of the similarity of physical and chemical properties of the isomers, it is difficult to accurate recognition of D-Trp from L-Trp. A series of technologies have been developed for the sensitive and accurate discrimination of chiral AAs.10-14 Unfortunately, circular dichroism shows low sensitivity. Instrumental analysis requires expensive equipment and expensive analysis cost. Surface enhanced Raman scattering depends on the types and shapes of substrates. Fluorescence detection is susceptible to interference from other substances, resulting in fluorescence quenching. These methods are cumbersome and time-consuming for the detection. Alternatively, electrochemical method is becoming increasingly an important method for the chiral recognition attributing to the high stability and sensitivity, low cost, and rapid detection.15-18 Although several strategies of electrochemical assay had been proposed over the past decades, most researchers focused on the selection of chiral selectors in the construction of electrochemical chiral sensors, including chitosan, β-cyclodextrin, bovine serum albumin and so on.19-21 The modification of chiral selectors would hinder the electron conduction efficiency on the electrode, decreasing the electrochemical intensity. Hence, development of a sensitive electrochemical chiral sensing platform remains necessary in the aspect of chiral isomers detection Target-recognition-induced NPs aggregation made colorimetric assays to be a common strategy for the quantification of enantiomers.22 However, the major problem of colorimetric assays is its poor sensitivity. Colorimetric assays can be initiated on the surface of electrode.23 On one hand, electrochemical assay exhibits superior sensitivity than colorimetric assays. On the other hand, electroactive NP assemblies on electrodes can largely amplify the signal responses.24 2


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Converting liquid-phase colorimetric assays into electrochemical assays is tremendously valuable for the dual amplification of signals in the ultrasensitive quantification of chiral enantiomers. Electroactive NPs dependent electrochemical sensors have drawn enormous attention due to good catalytic ability, high electron conductivity and favorable biological microenvironment.25 Electroactive NPs can produce significant DPV peaks due to ease of oxidation. In particular, considerable attention had been paid to single metal NPs (Ag NPs, Cu NPs, Zn NPs, Pd NPs ect).23,24,

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Compared with the single NPs, core-shell (CS) NPs possess improved stability and

amplified electroactive properties, such as Au@Cu2O NPs and Au@Ag NPs.27-31 Electroactive CS NPs dependent electrochemical chiral sensors have not been developed for the Trp isomer recognition. It was reported that the binding constant of NPs and L-Trp was smaller than that of NPs and D-Trp. The adsorbed mass of D-Trp and Cu2+ was larger than that of L-Trp and Cu2+.22, 32 In this assay, an intuitive liquid-phase approach is developed for the chiral Trp enantiomers recognition through the aggregations of Au NPs and Au@Ag NPs with the aid of Cu2+. The excellent electroactive properties promote Au@Ag NPs dependent colorimetric assays to convert onto electrode surface for the Trp enantiomers recognition. With the increasing amounts of D-Trp, Au@Ag NPs are assembled to dimers, trimmers and even network architecture, generating amplified DPV signals. The LOD for D-Trp is as low as 1.21 pM. This proposed electrochemical chiral sensor also achieves the accurate quantitative analysis of enantiomeric excess. Converting colorimetric chiral method into electrode surface not only improves the sensitivity but simplifies manipulation procedures. This sensing platform has great potential prospects in the aspect of enantioselective recognition of chiral isomers. EXPERIMENTAL SECTION Materials and Reagents.

Trisodium citrate, silver nitrate (AgNO3), chloroauric acid

(HAuCl4·4H2O) were bought from Sigma Chemical Co. (St. Louis, MO, USA). L/D-Trp, L/D-Pro , L/D-Cys, L/D-Ala , L/D-Ser and 4-aminobenzenesulfonic acid (4-ABSA) were gotten from Sinopharm Chemical Reagent Beijing Co. K2HPO4·3H2O and KH2PO4·3H2O were acquired from Shanghai Chemical Reagent Company. Ultrapure water was used in all experiments. Instrumentation. The pictures were recorded by a camera. UV-vis absorption spectra were measured through UV/Vis spectrophotometer (Model TU-1901). The shape of NPs was measured 3



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by a JEM-2100 TEM. Electrochemical curves were recorded on a CHI-650D electrochemical workstation (Shanghai CH Instruments Co., China). Synthesis and Modification of Au NPs. Au NPs were obtained reference to our previously reported article.33-34 The average diameter was 16.0 ± 1.5 nm. 100 µL 10 nM Au NPs was mixed with 100 µL ultrapure water and 300 µL Britton–Robinson (BR) buffer (pH 4.0).

An amount of

50 µL D-Trp (0.4 µM) was mixed with the mixture solution. Finally, the mixtures solution was reacted for 10 min and centrifuged to remove the excess D-Trp. Au NPs/D-Trp were redispersed in 50 µL water. Preparation of Au@Ag NPs. Au@Ag NPs were obtained by the reducing Ag+ on the surface of Au NPs.35 First, 30 mL 1 nM Au NPs was heated under stirring, and then 2.5 mL C6H5Na3O7·2H2O and 13.5 mM AgNO3 solution were simultaneously injected drop-wise into the boiled mixture for 10 min. And then,the color of the solution was changed, and the mixtures were heated. After 15 min, the temperature of colloidal solution is lowered to room temperature, and then was centrifuged and dispersed in 3 mL water. Four types of Au@Ag NPs were obtained by controlling the amounts of AgNO3 solution of 200, 400, 800, 1600 µL. Colorimetric Chiral Discrimination of Trp Enantiomers. Colorimetric chiral recognition was implemented by NPs . An aliquot of 100 µL ultrapure water and 100 µL 10 nM Au NPs were added into 300 µL BR buffer in an eppendorf tube. An amount of 50 µL D-Trp or L-Trp with different amounts (involving 0, 0.05, 0.1, 0.2, 0.4, 0.5, 1.0 and 5.0 µM) was injected into the above mixture and incubated for 5 min at 25 oC, respectively. And then, 50 µL 1 mM Cu2+ was added to the above solution. The mixtures were further incubated for 10 min. The color of solution was changed, and the UV-vis spectra were recorded. Au NPs dependent colorimetric chiral recognition was performed for the discrimination of Cys, Ala, Pro and Ser enantiomers. Analogously, 100 µL 10 nM Au@Ag NPs and 100 µL ultra-pure water were added into 200 µL BR buffer. An amount of 100 µL D-Trp or L-Trp with different concentrations (involving 0, 0.1, 0.3, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mM) was injected into the above mixture solution and reacted at 25oC, respectively. After 10 min, 50 µL Cu2+ was injected. Next,the mixtures solution was incubated for 20 min, and UV-vis spectra were recorded.

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Preparation and Modification of Glassy Carbon Electrode (GCE). GCE was polished using 0.3 µm and 0.05 µm alumina powders, and then it was washed by ethanol and double distilled water, respectively. 20 mM 4-ABSA was reduced for 30 min via a three-electrode system consisting of GCE, Pt wire and saturated calomel electrode, in order to modify sulfuric groups on GCE. The sulfonic groups were activated for 30 min by 40 mM PCl5 solution.36 Next, the functionalized GCE was rinsed with water and dried. 10 µL Au NPs/D-Trp was dripped on the as-modified GCE surface, and the as-modified GCE was rinsed by water. Fabrication of Au@Ag NP Assemblies Depended Electrochemical Chiral Sensors. An amount of 100 µL 10 nM Au@Ag NPs and 100 µL ultrapure water were added into 200 µL BR buffer. An amount of 100 µL D-Trp or L-Trp with different amounts (1, 5, 10, 50, 100, 300, 500, 800, 1000 pM) was mixed with the above solution. After 10 min, 50 µL 1 mM Cu2+ was added. The mixture solution was incubated for 20 min, and then centrifuged to remove the excess D-Trp. The concentration of Au@Ag NPs/Trp was 5 nM after adding water to 200 µL. An amount of 5 µL 1 mM Cu2+ was dripped on the Au NPs/D-Trp functionalized GCE. And thenr incubation for 30 min, GCE was rinsed with water, so as to remove the excess Cu2+. Then, as-prepared GCE was immersed in the above prepared Au@Ag NPs/Trp solution for 10 min, and then 50 µL 1 mM Cu2+ was mixed with the above solution. After 30 min, the modified electrode was washed three times. DPV of the as-prepared GCE was measured scanning from -0.4 to 0.6 V. The step potential was 0.4 mV·S-1. Electrochemical Chiral Recognition of Enantiomeric Excess. Different percent of D-Trp, involving 0%, 10%, 20%, 40%, 60%, 80% and 100%, were prepared by controlling the total concentration of D-/L-Trp at 1 nM. Trp was firstly reacted with Au@Ag NPs according to the above experimental procedures. And then Au NPs/D-Trp modified GCE was immersed in the Au@Ag NPs/Trp isomers solution containing 50 µL 1mM Cu2+ for 30 min. As-prepared GCE was rinsed three times and the DPV responses were recorded. The fabricated electrochemical chiral sensor was also performed in different percent of D-Trp (involving 10%, 30%, 50%, 70% and 90%) when the total concentrations of D-/L-Trp were 50, 100, 300, 500, 1000 pM, respectively. Specificity and Universal Application. The specificity of electrochemical methods was assessed with addition of 50 nM different AAs including L/D-Cys, L/D-Ala, L/D-Ser, L/D-Pro. 5



Au@Ag NPs was firstly reacted with these AAs in the same ways. Au NPs/D-Trp modified GCE was immersed in the above prepared Au@Ag NPs/AAs solution containing 50 µL 1mM Cu2+ for 30 min. The DPV responses of the as-prepared GCE were measured. This proposed electrochemical assay was also applied for the chiral discrimination of other AAs enantiomers (Cys, Ala, Ser and Pro) by changing the identification unit on the electrode from Au NPs/D-Trp to other D-AAs functionalized Au NPs. 100 µL 10 nM Au NPs was injected into 100 µL ultrapure water and 300 µL pH 4.0 BR buffer, and then 50 µL 0.4 µM D-Cys, D-Ala, D-Ser and D-Pro was injected to the mixture, respectively. The mixtures were incubated for 10 min and centrifuged to remove the excess D-AAs. Au NPs/D-AAs were redispersed in 50 µL water, and then were used for the modification of GCE. As-prepared GCE was applied for the chiral discrimination of 1 nM Cys, Ala, Ser and Pro enantiomers, respectively. RESULTS AND DISCUSSION Principle of Electroactive Au@Ag NP Assemblies Driven Signal Amplification for Ultrasensitive Detection D-/L-Trp. Firstly, D-Trp functionalized Au NPs were prepared and modified on the GCE surface, in order to capture D-Trp with the aid of Cu2+. Second, detected Trp molecules, were reacted with Au@Ag NPs. The larger binding constant between NPs and D-Trp enabled more D-Trp to load on the surface of NPs, in comparison to L-Trp (Figure S1A). Two D-Trp molecules can be connected by the chelation between –COOH, Cu2+ and nitrogen atom of indole ring and the large adsorbed mass of D-Trp and Cu2+. Au@Ag NPs were assembled on the electrode (Figure S1B).24,37 On one hand, more D-Trp existed, more D-Trp were modified on Au@Ag NPs, more complex Au@Ag NPs network assemblies were obtained, and stronger DPV signals appeared owing to one electron transfer from Ag0 to Ag+ (Scheme 1). In comparison to D-Trp, a small number of L-Trp was modified on Au@Ag NPs, and only Au@Ag NPs oligomers were assembled on the surface of electrode owing to the weaker adsorbed mass of Cu2+ and L-Trp, generating a weak DPV signal. A sensitive and accurate electrochemical chiral sensor was proposed for the quantification of D-/L-Trp ascribing to the larger binding constant between NPs, D-Trp and Cu2+, as well as the electroactive Au@Ag NP assemblies dependent signal accumulation.

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Synthesis and Characterization of NPs. Au@Ag NPs were synthesized by deposition of Ag shell on the surface of 16.0 ± 1.5 nm Au NPs (Figure 1A and Figure S2A). As demonstrated in Figure 1B, Au@Ag NPs exhibited uniform core-shell morphology and well disparity. The mean particle size of Au@Ag NPs was 19.2 ± 2.3 nm, and the shell thickness was 1.6 ± 0.9 nm (Figure 1C). The hydrodynamic diameters increased from 39.6 nm to 42.3 nm after the deposition of Ag shell (Figure S2B). Au NPs after Ag shell deposition not only showed blue-shifted LSPR which ranged from 520 nm to 495 nm, and also exhibited an acromion at 400 nm belonged to Ag shell. In comparison to the red color of Au NPs, we could clearly find that the color of Au@Ag NPs was orange (Figure 1D). Visual Discrimination of Trp isomers. The visual chiral discrimination of Trp isomers was performed by using NPs-Trp as sensing units and Cu2+ as cross-linking reagents. Au NPs would aggregate together with the increasing concentration of D-Trp. In comparison to the blank group, there were no obvious changes for the UV-vis spectrum of Au NPs while the concentration of D-Trp was lower than 0.4 µM. Only one plasmonic peak for Au NPs occurred at 520 nm, and the color of Au NPs was still red. With the increasing amounts of D-Trp from 0.5 µM to 5 µM, the peak at 520 nm gradually weakens, in addition, a new peak appeared at 609 nm and undergone a progressively stronger red shift by 89 nm and 196 nm (Figure 2A). The ratios of two peaks (I/I0) increased from 0.425 to 1.466 (I0 was the extinction intensity of Au NPs at 520, and I referred to the new extinction intensity of Au NPs in the presence of D-Trp). The color changed from red to blue. In comparison to L-Trp, the peaks at 520 nm and the color of Au NPs solution exhibited no any changes when the concentration of L-Trp reached up to 5 µM (Figure 2B). The chiral recognition can be achieved due to three reasons: (1) the larger binding constant between D-Trp and NPs (Figure S1A);24 (2) the chelation between –COOH, Cu2+ and nitrogen atom of indole ring of D-Trp (Figure S1B);24 (3) the larger adsorbed mass of D-Trp and Cu2+ than that of L-Trp and Cu2+ .37 To conduct the aforesaid strategy, colorimetric chiral discrimination of Trp was also performed by using Au@Ag NPs-Trp as sensing units and Cu2+ as cross-linking reagents. As illustrated in Figure 2C, no obvious changes at 400 nm were observed within 0.5 mM D-Trp. When the concentration of D-Trp ranged from 1 mM to 3 mM, a new peak at 568 nm appeared 7



due to the generation of Au@Ag NP dimers, trimmers, etc. When the concentration of D-Trp increased from 4 mM to 5 mM, an obvious extinction peaks at 694 nm appeared and red-shifted to 717 nm ascribing to the generation of Au@Ag NP network assemblies. The color varied to atrovirens from orange. It was demonstrated that D-Trp would immediately cause the agglomeration of Au@Ag NPs. Meanwhile, there was no distinct effect on Au@Ag NPs after the introduction of L-Trp when the amounts reached up to 5 mM (Figure 2D). The color was still orange. TEM images were applied for the characterization of Au@Ag NP assemblies at different concentration of Trp. As demonstrated in Figure 3A-D, Au@Ag NPs were assembled to dimers, trimmers, tetramers, oligomers and finally to networks with the increased amounts of D-Trp ranging from 2 to 5 mM. In sharp contrast to D-Trp, only Au@Ag NP dimers and trimmers were assembled when the concentration of L-Trp reached up to 5 mM, and there were almost no obvious differences for the Au@Ag NP assemblies in the range of 2 to 4 mM L-Trp (Figure 3E-H). The different color changes of NPs between L-Trp and D-Trp indicated the potential applications for the chiral identification of Trp isomers. Au NPs showed higher sensitivity for the colorimetric chiral discrimination of Trp than Au@Ag NPs. This was because that the clear color turn blue can be easily watched by naked eyes, in comparison to the unawareness color changes from orange to atrovirens. To further improve the sensitivity, the proposed strategy was transferred to electrode surfaces by taking advantages of electroactive properties of NP assemblies. Alternatively, Au@Ag NPs exhibited accurate and amplified DPV peaks at 0.2 V attributing to the oxidation progress from Ag0 to Ag+ on Ag shell. In sharp contrast to the electroactivity of Au@Ag NPs, stable Au NPs could not be easily oxidized to produce DPV peaks. Therefore, Au@Ag NPs were used as electroactive labels for the establishment of electrochemical chiral sensor. Electrochemical Performances of the Modified GCE. Au@Ag NPs exhibited stronger DPV signals than Ag NPs and Au NPs (Figure S3). The DPV responses of Au@Ag NPs were tunable by adjusting the thickness of Ag shell. As demonstrated in Figure S4, DPV intensity at 0.2 V for Au@Ag NPs increased when the amounts of AgNO3 solution changed from 200, 400, 800 to 1600 µL. Considering the large particle size may limit the assembly of the material, Au@Ag NPs by 8


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deposition of 800 µL AgNO3 solution was chosen as the electrochemical beacon. Au NPs were functionalized by D-Trp and then used for the modification of GCE. Au NPs/D-Trp functionalized GCE could capture D-Trp modified Au@Ag NPs using Cu2+ as a bridge. The assembled procedure step by step on the electrode surface was measured by electrochemical impedance spectroscopy (EIS). The impedance data was imitated using electrical equivalent circuit models in Z Simp Winsoftware as shown in Figure 4A, the electron transfer kinetics of [Fe(CN)6]3-/4- was rendered at distinct functionalized electrodes. Cdl was a double-layer electric capacity and Ret represented the transfer resistance of electrons from the liquid phase to the electrode. The diameter of the semicircle increased with the value of Ret. The warburg element (Zw) and electrolyte resistance (Rs) imply the diffusion of redox probe and bulk nature of the electrolyte solution. As demonstrated in Figure 4A, GCE after the deposition of ABSA showed a slight increased Ret due to the hindered electron transfer by ABSA (curve a-b). When the electrode was coated by Au NPs/D-Trp, the NPs film would be constructed on the electrode, hindering the electron transfer and increasing the value of Ret (curve c). Finally, the diffusion process of [Fe(CN)6]3-/4- was further prevented owing to the assemblies of Au@Ag NPs on the electrode. Importantly, D-Trp exhibited higher binding constant to NPs and Cu2+ than L-Trp. In comparison to Au@Ag NPs/L-Trp, more Au@Ag NPs/D-Trp would be assembled on the surface of electrode, resulting in higher Ret of the electrodes (curve d-e). The electrochemical performances of as-prepared electrodes were characterized in Figure 4B. No any electrochemical responses were observed for bare GCE, GCE/ABSA and Au NPs/D-Trp functionalized GCE/ABSA due to the stable states of Au0 (curve a-c). However, when D-Trp modified Au@Ag NPs were assembled on GCE, an obvious DPV peak appeared at 0.2 V, owing to one electron transfer from Ag0 to Ag+ (curve d). As demonstrated in SEM images of Figure S5, there was no any NPs on the surface of bare GCE, but D-Trp modified Au@Ag NP assemblies were formed on the surface of GCE. However, there was only a weak DPV peak when L-Trp modified Au@Ag NPs captured on the as-prepared GCE surfaces, due to the weak combination between L-Trp on Au@Ag NPs and Cu2+ (curve e). Interestingly, the peak currents enhanced with the increasing concentration of Cu2+. This was attributed to the formation of D-Trp-Au@Ag NP network assemblies using Cu2+ as cross-linking reagents for the signal accumulation. As 9



illustrated in Figure 4C, DPV peak at 0.2 V enhanced with the increasing amounts of Cu2+ ranging from 0 to 1 mM, and was almost no changes between 1 mM and 2 mM Cu2+. The DPV intensity of D-Trp-Au@Ag NP network assemblies in the presence of 1 mM Cu2+ was 1.5-fold higher than that of blank groups. The distinct and amplified DPV responses enabled the proposed Au@Ag NP assemblies depended electrochemical chiral sensors for the accurate and sensitive quantification of D-/L-Trp. Establishment of Electrochemical Chiral Transducer for Quantitative Discrimination of Trp Isomer. More D-Trp existed, more D-Trp-Au@Ag NPs would combine on electrodes, resulting in the assembly of D-Trp-Au@Ag NP network and generating strong DPV signal. As illustrated in Figure 5A, DPV intensity for Au@Ag NP assemblies became strong at 0.2 V with the increase of

D-Trp concentration

(1 pM - 1 nM). The calibration curves demonstrated a

well linear relationship of the current intensity and the amounts of D-Trp ranging from 5 pM to 1 nM. The equation was I (µA) = 0.00947 [CD-Trp/pM] + 0.54813, R2=0.99246 (Figure 5B). The LOD was 1.21 pM for D-Trp. Meanwhile, there were almost no any responses for electrochemical chiral sensor when the same concentration of L-Trp was used (Figure 5C-D). The electrochemical chiral sensor was performed for the detection of 1 nM D-Trp by following the same procedure every day. As demonstrated in Figure S6, it was found that the current peak did not change much each time. This developed electrochemical chiral sensor showed good reproducibility. Compared with colorimetric assay and

reported electrochemical chiral sensors, electroactive

Au@Ag NP assemblies driven signal amplification achieved high sensitivity of the electrochemical chiral discrimination of L-/D-Trp (Table 1). Electrochemical Determination of Enantiomeric Excess (ee) Values. The proposed electrochemical chiral sensor was employed to

measure the ee values. As illustrated in Figure

6A, the peak currents at 0.2 V increased with the increasing percentage of D-Trp from 10% to 100% when the total amount of Trp was 1 nM. The linear curves between peak current and percentage of D-Trp was obtained as shown in Figure 6B. The equation was I (µA) = 0.09861[D-Trp%] + 0.57059, R2=0.99173 (Figure 6B). To further prove the feasibility of the established assay, the selective recognition and quantification of D-Trp from L-Trp was performed at various proportion of D-Trp in different total concentration of Trp enantiomers, involving 50, 100, 300, 500, 800 and 10


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1000 pM, respectively. As illustrated in Figure 6C, the peak current enhanced when the proportion of D-Trp increased. For an unknown sample, the DPV intensity at 0.2 V can be obtained by this developed electrochemical chiral sensor. According to the 3D plots of different proportion of D-Trp and the peak currents in Figure 6C, the ee values can be calculated. In comparison to traditional HPLC, this developed electrochemical chiral sensor is simple and sensitive, and do not requires expensive equipment and expensive analysis cost. Selectivity Evaluation and Universal Application. The selectivity of the proposed electrochemical chiral sensor was evaluated with addition of other chiral enantiomers, involving L-/D-Cys, L-/D-Ala, L-/D-Ser, L-/D-Pro. As demonstrated in Figure 6D, high current intensity was merely observed in the being of D-Trp. The modification of Au NPs/D-Trp on the electrode was used for the selective detection of D-Trp, owing to the affinity between D-Trp, NPs and Cu2+. the peak currents of other AAs and blank groups had no obvious changes . Therefore, this proposed electrochemical chiral sensor shows potential prospects for the selective recognition of Trp enantiomers through Au NPs/D-Trp as the identification units on the electrode. Not only D-Trp can induce the aggregation of NPs, other D-AAs (such as Cys, Ala, Ser, Pro) can also result in the assembly of NPs. As demonstrated in Figure S7, D-Cys, D-Ala, D-Ser and D-Pro exhibited high affinity to Au NPs than L-Cys, L-Ala, L-Pro and L-Ser, resulting in the aggregation of NPs and obvious SPR changes.39 Importantly, this proposed electrochemical chiral sensor can also be applied for the chiral recognition of these D-AAs, by changing the identification unit on the electrode from Au NPs/D-Trp to other D-AAs functionalized Au NPs. As illustrated in Figure 7A, the electrochemical responses of chiral sensor to D-AAs were different from that of L-AAs. This developed electrochemical chiral sensor achieved high sensitivity and universal application for the recognition of these D-AAs, in comparison to colorimetric approach and UV-vis spectrometer. Even though high electrochemical currents were observed for Cys enantiomers, the responses between D-Cys and L-Cys were rather small, due to the strong covalent bond between -SH of Cys and metal NPs. Among these AAs, D-Trp showed a significantly strongest current, and Trp enantiomers showed highest current ratios than other AAs. The ratio of current intensity between D-Trp and L-Trp was 18.42, 5.25, 8.75, 8.5-fold higher than that for Cys, Ala, Ser and Pro enantiomers. The different sizes of the side chains for different AAs 11



were responsible for the different aggregation trends observed.37 Thus, the proposed electrochemical chiral sensor showed universal application for the identification of D-Trp, D-Cys, D-Ala, D-Pro and D-Ser by designing the identification unit on the electrode, but was more attractive for the chiral identification of Trp isomer with high sensitivity. CONCLUSIONS In summary, we propose an electrochemical chiral sensor for the ultrasensitive chiral identification of Trp isomer. In comparison to L-Trp, D-Trp causes the aggregations of NPs, resulting in the color changes. D-Trp induces the assembly of electroactive Au@Ag NP networks on the electrode with the aid of Cu2+, generating strong DPV signals. L-Trp results in the assembly of Au@Ag NP oligomers with low DPV signals. The different geometry of Au@Ag NP assemblies determined by D-Trp and L-Trp produces distinct DPV signals. By converting colorimetric assay onto electrode surface and taking advantages of target-induced electroactive NP assemblies, dual amplified signal responses are obtained in the proposed electrochemical chiral sensing platform for the ultrasensitive assay of D-Trp and enantiomeric excess. It is promising to construct an electrochemical chiral sensing platform by converting from colorimetric assay and utilizing the excellent electrochemical properties of nanoscale electroactive CS NPs. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssusche-XXXX. The statistical analysis of the diameter of Au NPs and the DLS analysis of NPs, UV-vis spectra of Au NPs dependent visual chiral recognition of other AAs, the SEM image of bare GCE and the modified GCE, the DPV curves of Au NPs, Ag NPs and Au@Ag NPs modified-GCE, and the DPV plots of Au@Ag NPs with different Ag-shell modified-GCE were provided. AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Notes The authors declare no competing financial interest. 12


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ACKNOWLEDGMENTS This work is financially supported by the National Key Research and Development Program of China (2017YFC1601706), National First-class Discipline Program of Food Science and Technology (JUFSTR20180302), Natural Science Foundation of Jiangsu Province (BK20171136), the foundation of Changzhou Key Lab of New Textile Materials (No. 201701) and the 111 Project (B13025). REFERENCES (1) Gao, X.; Zhang, X.; Deng, K.; Han, B.; Zhao, L.; Wu, M.; Shi, L.; Lv, J.; Tang, Z. Excitonic Circular Dichroism of Chiral Quantum Rods. J. Am. Chem. Soc. 2017, 139, 8734−8739, DOI: 10.1021/jacs.7b04224. (2) Chan, C. W.; Laurini, E.; Posocco, P.; Pricl, S.; Smith, D. K. Chiral recognition at self-assembled multivalent (SAMul) nanoscale interfaces - enantioselectivity in polyanion binding. Chem Commun (Camb) 2016, 52 (69), 10540-10543, DOI: 10.1039/c6cc04470k. (3) Zhu, Y.; Wang, H.; Wan, K.; Guo, J.; He, C.; Yu, Y.; Zhao, Y.; Zhang, Y.; Lv, J.; Shi, L.; Zhang, X.; Shi, X.; Tang, Z. Enantioseparation of Au20(PP3)4Cl4Angewandte Chemie Clusters with Intrinsically Chiral Cores. Angew. Chem. Int. Ed. 2018, 57, 9059-9063, DOI:10.1002/anie.201805695. (4) Gao, X.; Zhang, X.; Zhao, L.; Huang, P.; Han, B.; Lv, J.; Qiu, X.; Wei, S. H.; Tang, Z. Y. Distinct Excitonic Circular Dichroism between Wurtzite and Zincblende CdSe Nanoplatelets. Nano Lett. 2018, 18, 6665-6671, DOI: 10.1021/acs.nanolett.8b01001. (5) Wu, D.; Yu, Y.; Zhang, J.; Guo, L.; Kong, Y. Chiral Poly(ionic liquid) with Nonconjugated Backbone as a Fluorescent Enantioselective Sensor for Phenylalaninol and Tryptophan. ACS applied materials & interfaces 2018, 10 (27), 23362-23368, DOI: 10.1021/acsami.8b04869. (6) Kimura, T.; Hamase, K.; Miyoshi, Y.; Yamamoto, R.; Yasuda, K.; Mita, M.; Rakugi, H.; Hayashi, T.; Isaka, Y. Chiral amino acid metabolomics for novel biomarker screening in the prognosis of chronic kidney disease. Sci. Rep. 2016, 6 (1), 26137, DOI: 10.1038/srep26137 (7) Zhang, Z.; Liu, Y.; Liu, P.; Yang, L.; Jiang, X.; Luo, D.; Yang, D. Non-invasive detection of gastric cancer relevant d-amino acids with luminescent DNA/silver nanoclusters. Nanoscale 2017, 9 (48), 19367-19373, DOI: 10.1039/c7nr07337b. (8) Yongxin, T.; Jiangying, D.; Yong, K.; Yan, S. Temperature-sensitive electrochemical recognition of tryptophan enantiomers based on β-cyclodextrin self-assembled on poly(L-glutamic acid). Anal. Chem. 2014, 86 (5), 2633-2639, DOI: 10.1021/ac403935s. (9) García-Carmona, L.; Moreno-Guzmán, M.; González, M. C.; Escarpa, A. Class enzyme-based motors for “on the

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Captions：

Scheme 1. Scheme of electroactive Au@Ag NP assemblies driven signal amplification for ultrasensitive chiral discriminition of D-/L-Trp.

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Figure 1. Representative TEM images of (A) Au NPs, (B) Au@Ag NPs. Insert in B, the enlarged TEM images. The scale bar was 20 nm. (C) Statistical analysis of the diameters of Au@Ag NPs. Insert, statistical analysis of the thickness of Ag shell. (D) UV−vis spectrum of Au NPs and Au@Ag NPs. Insert, the photographs of Au@Ag NPs (left) and Au NPs (right).

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Figure 2. (A-B) UV−vis spectrum of Au NPs dependent visual chiral recognition of (A) D-Trp and (B) L-Trp. (C-D) UV−vis spectrum of Au@Ag NPs dependent visual chiral identification of (C) D-Trp and (D) L-Trp. Insert, the corresponding photographs of Au NPs and Au@Ag NPs solution in the existence of different concentration of L-Trp and D-Trp.

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Figure 3. (A-D) TEM images of Au@Ag NP assemblies in the existence of 2, 3, 4 and 5 mM D-Trp. (E-H) TEM images of Au@Ag NP assemblies in the existence of 2, 3, 4 and 5 mM L-Trp.

19



Figure 4. (A) EIS of electrodes in 5 mM [Fe(CN)6]3-/4- solution. (a) GCE, (b) GCE/ABSA, (c) GCE/ABSA + Au NPs/D-Trp, (d) GCE/ABSA + Au NPs/D-Trp + Au@Ag NPs/D-Trp, (e) GCE/ABSA + Au NPs/D-Trp + Au@Ag NPs/L-Trp. (B) DPV curves of different functionalized electrodes in PBS (pH 7.4) solution. (a) GCE, (b) GCE/ABSA, (c) GCE/ABSA +Au NPs/D-Trp, (d) GCE/ABSA +Au NPs/D-Trp + Au@Ag NPs/D-Trp, (e) GCE/ABSA + Au NPs/D-Trp + Au@Ag NPs/L-Trp. (C) DPV curves of the electrochemical chiral sensor in the existence of different amounts of Cu2+.

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Figure 5. (A) DPV curves of the fabricated electrochemical chiral sensing platform at different amounts of D-Trp. (B) The standard curves between the amounts of D-Trp and the peak currents at 0.2 V. (C) DPV curves of theelectrochemical chiral sensing platform with different amounts of L-Trp. (D) The curves between the amounts of L-Trp and the peak currents at 0.2 V.

21



Figure 6. (A) DPV plots of the electrochemical chiral sensing platform for the detection of different proportion of D-Trp when the total concentration of Trp was 1 nM. (B) Calibration curve of peak currents at 0.2 V versus the proportion of D-Trp. (C) 3D plots of different proportion of D-Trp (10%, 30%, 50%, 70% and 90%) and the peak currents when the different total concentrations of Trp were 50, 100, 300, 500 and 1000 pM, respectively. (D) The selectivity of the electrochemical chiral sensing platform for the recognition of Trp enantiomers and other AAs.

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Figure 7. (A) DPV responses of the electrochemical chiral sensing platform for the chiral discrimination of L/D-Trp, L/D-Cys, L/L-Ala, L/D-Ser, L/D-Pro. (B) The current intensity at 0.2 V and ΔI of the electrochemical chiral sensor in the presence of different AAs. ΔI is the ratio of current intensity of peaks at 0.2 V in the presence of D-type and L-type AAs.

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Table 1. Comparison of chiral discrimintion of L-/D-Trp by different electrochemical chiral sensors. References

Methods

Linear range (M)

LODs

8

P-L-Glu/β-CD/GCE

Qualitative analysis

/

21

ae-CS/Cu-β-CD/GCE


/

38

anionic-/cationic-pillar arenes multilayer films

1.0×10-6-3.0×10-4

0.33×10-6 M (L-Trp)

39

Cu2-β-CD/P-L-Glu/G CE


/

40

SCS/GCE


/

41

Thi-GR

5×10-7-2.5×10-3

1.7×10-7 M

This work

Electroactive Au@Ag NP assemblies

5.0×10-12-1.0×10-9

1.21 ×10-12 M

24


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TOC Figure A chiral electrochemical transducer is proposed for the tryptophan isomers detection by using electroactive Au@Ag NPs as electrochemical tags.

25


Electroactive Au@Ag NP Assemblies Driven Signal Amplification for

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