An efficient chiral sensing platform based on graphene quantum dot–tartaric acid hybrids

Abstract

Graphene quantum dots (GQDs) and tartaric acid (TA) were successively electrodeposited onto the surface of a glassy carbon electrode (GCE). Due to the intrinsic chirality of L-(+)-TA and D-(−)-TA and the signal amplification function of GQDs, the GQDs–TA hybrid modified GCE could be regarded as an efficient electrochemical chiral sensing platform and used for chiral recognition of tryptophan (Trp) isomers. The recognition efficiency with the proposed GQDs–TA is significantly higher than that with TA alone. Of particular interest, GQDs-L-(+)–TA and GQDs-D-(−)–TA exhibit completely opposite selectivity toward L-Trp and D-Trp. Also in this work, the pH-sensitive enantiorecognition of the GQDs–TA hybrids was investigated.

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Introduction

L-Tryptophan (L-Trp) is an essential amino acid since it plays vital roles in all life forms and is an important constituent of most proteins and a precursor of the neurotransmitter serotonin.¹ D-Trp has quite similar physicochemical properties to L-Trp, however, D-Trp, a nonprotein amino acid, does not take part in the metabolism pathways of a living system.² As a result, the chiral recognition of Trp isomers has become extraordinarily important. Among the techniques employed for discrimination of Trp isomers, electrochemistry has drawn considerable attention owing to the advantages of easy operation, low cost, high accuracy and sensitivity.^3–7

Tartaric acid (TA), a kind of natural organic acid, possesses two asymmetric carbon atoms, and thus there exist three isomers of TA, i.e., optically active L-(+)-TA, D-(−)-TA and optically inactive meso-TA. The chirality of L-(+)-TA has been explored for separation of chiral compounds in high-speed countercurrent chromatography (HSCCC).^8,9 Also, copper(II) complexes of L-(+)-TA have been reported for resolution of racemic malic acid¹⁰ and a series of sympathomimetics¹¹ by ligand-exchange capillary electrophoresis (CE). However, as far as we are aware, there is no report on the electrochemical enantiorecognition of chiral compounds based on L-(+)-TA and/or D-(−)-TA, most likely due to the poor electrical conductivity of TA.

Recently, the electrochemistry of graphene and its derivatives has aroused increasing attention from the researchers in various fields,^12–15 owing to the fascinating features of large surface area, high conductivity and electron transfer rate, and excellent mechanical strength of graphene.^16,17 Among the derivatives of graphene, graphene quantum dots (GQDs) are a kind of zero-dimensional (0D) material with characteristics derived from both carbon dots and graphene.¹⁸ GQDs exhibit outstanding features due to edge effects and quantum confinement, which are similar to carbon dots,¹⁹ and have been applied in the field of electrochemical sensors.^20–22 However, little attention has been paid to GQDs-based chiral sensing, most likely due to the lack of chiral sites in GQDs. More recently, chiral recognition of tryptophan (Trp) isomers is successfully achieved in our group by using the composites of GQDs and natural polysaccharides (chitosan²³ and β-cyclodextrin²⁴) as the recognition materials, in which the two natural polysaccharides can provide chiral microenvironment needed for chiral recognition, opening a new avenue for the construction of electrochemical chiral sensing platform with GQDs-based composite materials.

In the present work, L-(+)-TA and D-(−)-TA are used as chiral reagents and hydrogen-bonded with GQDs, and the obtained GQDs–TA hybrids are applied for electrochemical recognition of Trp isomers for the first time. The GQDs–TA hybrids are capable of recognizing Trp isomers owing to the existence of optically active TA, on the other hand, GQDs in the hybrids can generate amplified recognition signals (peak current ratio) compared with TA alone. And therefore, the synergistic effects of GQDs and TA result in efficient enantiorecognition of Trp isomers. Interestingly, GQDs-L-(+)–TA and GQDs-D-(−)–TA exhibit completely opposite selectivity toward L-Trp and D-Trp. Finally, the pH-sensitive recognition with the GQDs–TA hybrids is also studied.

Experimental

Reagents and apparatus

L-(+)-TA, D-(−)-TA, L-Trp and D-Trp were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Citric acid monohydrate and all other reagents not mentioned were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All solutions were prepared with ultrapure water (18.2 MΩ). The Fourier transform infrared (FT-IR) spectra of different samples were recorded on a Nicolet FTIR-8400S spectrophotometer (Shimadzu, Japan), and the scanning electron microscopy (SEM) images of GQDs and GQDs-L-(+)–TA modified glassy carbone electrode (GCE, 3 mm in diameter) were recorded on a Supra55 field emission scanning electron microscope (Zeiss, Germany). The transmission electron microscopy (TEM) image of GQDs was recorded on a JEM-2100 transmission electron microscope (JEOL, Japan). X-ray photoelectron spectroscopy (XPS) was carried out on a Thermo Fisher ESCALAB 250Xi X-ray photoelectron spectrometer. All electrochemical experiments were performed on a CHI 660D electrochemical workstation (Beijing, China) in a three-electrode cell consisting of a platinum foil as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode, and modified GCE as the working electrode, respectively.

Fabrication of GQDs–TA modified GCE (GQDs–TA/GCE)

GQDs were fabricated according to a previously reported bottom-up pyrolysis method (200 °C, 30 min) using citric acid (2 g) as the precursors,²⁵ and the obtained GQDs were dissolved in ultrapure water under ultrasonic conditions. Next, a GCE was placed into the GQDs solution (2 mg mL⁻¹), and cyclic voltammetry (CV) was performed for the deposition of GQDs onto the GCE working electrode in the potential range between 0 and 1.0 V at a scan rate of 100 mV s⁻¹ for fifty cycles.²³ Finally, the GQDs modified GCE (GQDs/GCE) was transferred into 25 mL of 0.7 M L-(+)-TA aqueous solution (pH 3.56), and CV was carried out again for the electrodeposition of L-(+)-TA onto the GQDs/GCE in the potential range from 0 to 0.5 V at a scan rate of 100 mV s⁻¹ for thirty repeating cycles to fabricate the GQDs-L-(+)–TA/GCE. Since the pK_a value of TA is 3.04, it is negatively charged at pH 3.56 and can be prone to be electrodeposited at positive potentials due to electrostatic interactions. Similarly, GQDs-D-(−)–TA/GCE was fabricated using D-(−)-TA instead of L-(+)-TA in the second CV process. For a comparison, TA modified GCE (TA/GCE) was also fabricated with just the second CV, i.e., TA was directly electrodeposited onto the surface of GCE without the pre-deposited GQDs.

Electrochemical enantiorecognition of Trp isomers

L- and D-Trp were dissolved in 25 mL of 0.1 M acetate buffer solutions (pH 6.0), respectively (0.5 mM). Next, the three-electrode system using GQDs-L-(+)–TA/GCE or GQDs-D-(−)–TA/GCE as the working electrode was transferred into the solution of L-Trp or D-Trp, and the Trp isomers were hydrogen-bonded to the GQDs-L-(+)–TA/GCE or GQDs-D-(−)–TA/GCE, respectively. After 90 s, the system was transferred into 25 mL of 0.1 M acetate buffer solutions (pH 6.0), and the differential pulse voltammograms (DPVs) of L- and D-Trp bound to the working electrode were recorded from 0.4 to 1.0 V. The chiral recognition of Trp isomers was achieved successfully based on the discernable differences in the oxidation peak current of L- and D-Trp, and the whole process is illustrated in Fig. 1. The enantiorecognition efficiencies at different modified GCEs including GQDs/GCE, TA/GCE and GQDs–TA/GCE were evaluated based on the peak current ratio (I_L/I_D or I_D/I_L).

Fig. 1 Schematic illustration showing the chiral recognition of Trp isomers with GQDs-L-(+)–TA/GCE and GQDs-D-(−)–TA/GCE.

Results and discussion

Characterization of GQDs

TEM was employed to characterize the as-prepared GQDs (Fig. 2). It is obvious that the GQDs are highly dispersed with the diameter less than 4 nm, and the average size of the GQDs is determined to be about 2.4 nm.

Fig. 2 TEM image of GQDs. Inset: size distribution histograms of GQDs.

The composition of the produced GQDs was further characterized by XPS (Fig. 3). Fig. 3A indicates that oxygen and carbon are the dominant elements of the GQDs. Fig. 3B shows the resolved O 1s spectra of the GQDs, and the two fitted peaks at 532.1 and 533.3 eV are assigned to the C=O and C–OH/C–O–C groups, respectively,²⁶ clearly indicating the presence of oxygen-containing groups on the GQDs.

Fig. 3 (A) XPS survey spectra of GQDs. (B) Resolved O 1s spectra of GQDs.

Characterization of GQDs-L-(+)–TA

Fig. 4 shows the FT-IR spectra of GQDs-L-(+)–TA, GQDs and L-(+)-TA. For GQDs, the peaks at 1716 and 1633 cm⁻¹ are attributed to stretching vibrations of C=O and C=C, respectively.²⁷ For L-(+)-TA, the two characteristic peaks located at 3409 and 3334 cm⁻¹ are ascribed to the asymmetrical and symmetrical stretching vibrations of –OH groups,²⁸ and another two peaks at 1735 and 1255 cm⁻¹ are assigned to the stretching vibrations of C=O and the bending vibrations of C–O–H, respectively.²⁹ All these characteristic peaks can be observed on the FT-IR spectrum of GQDs-L-(+)–TA, suggesting the successful combination of GQDs and L-(+)-TA. It is noteworthy that the two peaks related to the –OH groups (3409 and 3334 cm⁻¹) in L-(+)-TA show a red shift to 3404 and 3332 cm⁻¹, respectively, indicating that the –OH groups in L-(+)-TA molecules are involved in the formation of hydrogen-bonding between L-(+)-TA and the oxygen-containing groups on GQDs.²⁸

Fig. 4 FT-IR spectra of GQDs-L-(+)–TA, GQDs and L-(+)-TA.

Fig. 5 shows the SEM images of GQDs/GCE and GQDs-L-(+)–TA/GCE obtained via a step-by-step electrodeposition. As can be seen, GQDs are well distributed on the surface of GCE after CV (Fig. 5A), which function as a scaffold for the subsequent deposition of L-(+)-TA since the oxygen-containing groups on GQDs could form hydrogen-bonding with the –OH groups in TA, as revealed by the FT-IR spectrum of the hybrids (Fig. 4). After the second CV, the hybrids of GQDs-L-(+)–TA are formed on the GCE, exhibiting a morphology quite different from that of GQDs/GCE (Fig. 5B).

Fig. 5 SEM images of GQDs/GCE (A) and GQDs-L-(+)–TA/GCE (B).

Electrochemical properties of GQDs-L-(+)–TA

The electrochemical properties of the GQDs-L-(+)–TA are studied. Fig. 6 shows the cyclic voltammograms of 10 mM Fe(CN)₆^3−/4− in 0.1 M KCl at bare GCE, L-(+)-TA/GCE, GQDs/GCE and GQDs-L-(+)–TA/GCE. A pair of well-defined redox peaks appears at the bare GCE, which is attributed to the redox between Fe(CN)₆³⁻ and Fe(CN)₆⁴⁻. The peak currents decrease a little at the L-(+)-TA/GCE due to the poor electrical conductivity of L-(+)-TA compared with GCE. It is noted that the peak currents are further decreased at the GQDs/GCE, and the phenomenon could be attributed to the fact that the electronegative oxygen-containing groups on GQDs hamper the electron transfer of Fe(CN)₆^3−/4− onto the electrode surface.³⁰ The lowest currents are observed at the GQDs-L-(+)–TA/GCE due to the coexistence of GQDs and L-(+)-TA in the hybrids.

Fig. 6 Cyclic voltammograms of 10 mM Fe(CN)₆^3−/4− in 0.1 M KCl at bare GCE (a), L-(+)-TA/GCE (b), GQDs/GCE (c) and GQDs-L-(+)–TA/GCE (d).

Electrochemical impedance spectroscopy (EIS) is adopted for further analysis of the GQDs-L-(+)–TA/GCE, since it can give direct information regarding the electrode-solution interfaces.³¹ Fig. 7 shows the Nyquist plots of 10 mM Fe(CN)₆^3−/4− in 0.1 M KCl at bare GCE, L-(+)-TA/GCE, GQDs/GCE and GQDs-L-(+)–TA/GCE. All the four plots consist of a suppressed semicircle at high frequency corresponding to the interfacial charge transfer resistance (R_ct) and a straight line at low frequency corresponding to the Warburg resistance (W_d). As can be seen, the EIS at the four electrodes exhibits the same tendency as that in the cyclic voltammograms (Fig. 6). That is, the GQDs-L-(+)–TA/GCE shows remarkably larger R_ct than bare GCE, GQDs/GCE and L-(+)-TA/GCE, suggesting that the resistance at the interfaces of GQDs-L-(+)–TA/GCE and the electrolyte is the highest.

Fig. 7 Nyquist plots of 10 mM Fe(CN)₆^3−/4− in 0.1 M KCl at bare GCE (a), L-(+)-TA/GCE (b), GQDs/GCE (c) and GQDs-L-(+)–TA/GCE (d). Inset: equivalent circuit (left) and magnified Nyquist plots at bare GCE and L-(+)-TA/GCE (right).

Electrochemical enantiorecognition of Trp isomers

The DPVs of L- and D-Trp bound to the GQDs-L-(+)–TA/GCE and the GQDs-D-(−)–TA/GCE at 20 °C are shown in Fig. 8. For a comparison, the DPVs of L- and D-Trp bound to GQDs/GCE, L-(+)-TA/GCE and D-(−)-TA/GCE are also recorded. As expected, the GQDs/GCE is incapable of recognizing Trp isomers as the peak current ratio is very close to 1.0 (Fig. 8A), which could be ascribed to the lack of chiral microenvironment in GQDs. After the introduction of optically active TA including L-(+)-TA and D-(−)-TA to the GCE surface, the peak current ratio is increased to some extent (I_D/I_L = 1.34 in Fig. 8B, I_L/I_D = 1.28 in Fig. 8C). The increased recognition efficiency is no doubt attributed to the existence of chiral sites derived from TA, since the optically active L-(+)-TA (or D-(−)-TA) can generate different steric hindrance during the combination between L-(+)-TA (or D-(−)-TA) and the Trp isomers. However, the recognition efficiency is still unsatisfactory, which might be due to the fact that the poor electrical conductivity of TA is disadvantageous to the electrodeposition of TA on the surface of GCE. It is exciting to find that the recognition efficiency is further increased at GQDs-L-(+)–TA/GCE and GQDs-D-(−)–TA/GCE (I_D/I_L = 2.47 in Fig. 8D, I_L/I_D = 2.71 in Fig. 8E). Obviously, both GQDs and TA play crucial roles in the chiral recognition of Trp isomers. On one hand, TA including L-(+)-TA and D-(−)-TA provides chiral sites for enantiorecognition; on the other hand, GQDs can act as a scaffold for the subsequent modification of TA to the recognition matrix through the formation of hydrogen-bonding, resulting in increased amount of TA to be electrodeposited on the surface of GQDs/GCE. Of particular interest, it is found that GQDs-L-(+)–TA (or L-(+)-TA) and GQDs-D-(−)–TA (or D-(−)-TA) exhibit completely opposite selectivity for L-Trp and D-Trp. That is, GQDs-L-(+)–TA and L-(+)-TA show higher affinity for D-Trp than L-Trp, leading to higher oxidation peak current of D-Trp. On the contrary, GQDs-D-(−)–TA and D-(−)-TA show higher affinity for L-Trp than D-Trp. Although the exact mechanisms are still unclear at the current stage, it can be concluded that the two optically active isomers of TA (Fig. 9) exhibit opposite affinity for the Trp isomers, probably due to the formation of stable hydrogen-bonds between L-(+)-TA and D-Trp, and between D-(−)-TA and L-Trp, as shown in Fig. 10.

Fig. 8 Differential pulse voltammograms of L- and D-Trp at GQDs/GCE (A), L-(+)-TA/GCE (B), D-(−)-TA/GCE (C), GQDs-L-(+)–TA/GCE (D) and GQDs-D-(−)–TA/GCE (E) in 0.1 M acetate buffer solution of pH 6.0.

Fig. 9 Two optically active isomers of tartaric acid.

Fig. 10 Possible combination between L-(+)-TA and D-Trp, and between D-(−)-TA and L-Trp through the formation of stable hydrogen-bonds.

pH-sensitive enantiorecognition with GQDs-L-(+)–TA

As shown in Fig. 11, the recognition efficiency with the GQDs-L-(+)–TA/GCE is strongly dependent on the acidity for the enrichment and determination of Trp isomers. The highest recognition efficiency is achieved in the pH range from 5.5 to 6.0, and it decreases with decreasing pH values (3.5–5.0). The low efficiency at low pH values is most likely due to the increased solubility of TA in strong acidic medium, leading to the instability of the GQDs-L-(+)–TA recognition systems. But, surprisingly, further increase in pH (6.5) does not improve but deteriorate the recognition efficiency. The isoelectric point of Trp is 5.89,³² and thus both L-Trp and D-Trp are negatively charged at pH 6.5. The electrostatic repulsion between negatively charged Trp isomers and the abundant oxygen-containing groups on GQDs would impair the combination of Trp isomers with the GQDs-L-(+)–TA hybrids, leading to decreased recognition efficiency with the GQDs-L-(+)–TA/GCE.

Fig. 11 Peak current ratio of D-Trp to L-Trp combined with GQDs-L-(+)–TA/GCE at different pH values. Error bars represent standard deviation for three independent measurements.

Conclusions

The hybrids of GQDs and L-(+)-TA (or D-(−)-TA) are facilely fabricated through a step-by-step electrodeposition method on GCE, and the obtained GQDs-L-(+)–TA/GCE and GQDs-D-(−)–TA/GCE are used for the construction of a pH-sensitive chiral sensing platform for the enantiorecognition of Trp isomers for the first time. The recognition efficiency at the proposed chiral interfaces is greatly higher than those at GQDs/GCE and L-(+)-TA/GCE (or D-(−)-TA/GCE) due to the synergistic effects of GQDs and optically active L-(+)-TA (or D-(−)-TA). Interestingly, GQDs-L-(+)–TA and GQDs-D-(−)–TA exhibit completely opposite selectivity for L-Trp and D-Trp, although the exact explanations are on the way. The findings of the present work might open up a new window for the construction of convenient and simple chiral interfaces for the electrochemical enantiorecognition of other chiral compounds.

Acknowledgements

The contributions of Yin Yu and Wenjie Liu were equal. The authors are grateful to the financial supports from Advanced Catalysis and Green Manufacturing Collaborative Innovation Center (ACGM2016-06-27) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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