Chiral detection using reusable fluorescent amylose-functionalized graphene

Abstract

By using DNA or a peptide as a common probe, graphene-based biosensing has made significant progress. However, to the best of our knowledge, a graphene-based chiral sensor has not been reported. Chiroselective recognition is perhaps the most subtle to achieve because of the similarity of the optical enantiomers. Therefore, besides using DNA or peptides as probes, developing graphene-based sensors with chiral selectivity is highly desirable. Here a reusable natural cheap polysaccharide, amylose-functionalized graphene was developed for highly sensitive and visual fluorescent chiral sensing. The detection sensitivity toward L-Trp is over 100-times higher than that of recently reported electrochemical sensors and colorimetric sensors. In comparison with commonly used DNA or peptides as a probe, natural amylose is more attractive because of its low cost, ready availability, simple manipulation and renewability. The specific selectivity for tryptophan (Trp) enantiomers towards other essential amino acids allows potential chiroselective analysis of Trp in complex samples such as biological fluids. This design can, in principle, be implemented for other slender target molecules that can form an inclusion complex with amylose.

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Introduction

Graphene (G), a one-atom-thick two-dimensional sheet of sp²-conjugated atomic carbon, has received much attention in recent years.¹ The unique two-dimensional (2D) nanostructure and the large specific surface area make G an excellent biosensor.^2–9 G has been demonstrated to be an efficient fluorescence quencher for organic fluorescent molecules.⁶ Both energy-transfer and electron-transfer processes have been proposed to be responsible for the deactivation of excited fluorophores on the G surface.^6,10 In combination with G properties and DNA aptamer, nucleic acids hybridization or peptide-protein specific interactions, significant progress on biosensing has been achieved by commonly used fluorescent dye-labeled DNA or peptides as the optical probe. However, to the best of our knowledge, there has been no report on G-based chiroselective sensing. Among all forms of molecular recognition, chiroselective recognition is perhaps the most subtle to achieve because of the similarity of the optical enantiomers.¹¹ Therefore, besides using DNA or peptide as probes, developing graphene-based sensors with chiral selectivity is highly desirable.

Amylose, a natural linear polysaccharide consisting of D-(+)-glucose units linked through (1 → 4)-α-glycosidic linkages, is a well-known host molecule that selectively forms inclusion complexes with slender guest molecules having relatively lower molecular weight by hydrophobic interaction between guests and the helical cavity of amylose.^12–14 It has been extensively applied in chiral capillary electrophoresis and chiral chromatography by us^15,16 and other laboratory,^17–19 supramolecular science,^20,21 and biomimic technology²² because of its optical activity and amphiphilicity.^23,24 Recently, amylose has even been used as dispersants for carbon nanotubes in aqueous solutions by disposing its hydrophobic methines towards the surface of carbon nanotubes and hydrophilic hydroxyls towards bulk solution.^25–27 Compared with other biomolecules, such as DNA and proteins, amylose is more attractive because of its low cost, ready availability, simple manipulation and renewability. However, so far, natural amylose has not been employed as a probe in a G-based detection platform. Based on amylose properties and its chiral selectivity, we report the first example of a G-based sensor with highly chiral selectivity.

Our strategy for the fabrication of a chiral sensor based on amylose and G is illustrated in Scheme 1. First, the fluorescent dye anthracene-labeled amylose (AA) is synthesized according to the procedure published by Hanashiro and Takeda.²⁸ Next, G is reduced from graphene oxide (rGO) and is non-covalently functionalized by AA to produce AA-rGO hybrids through the interactions between rGO^29,30 and amylose chains.^23,24 When AA is incubated with rGO, it undergoes stacking interactions with the largely hydrophobic basal plane of rGO via hydrophobic methines, and also forms H-bonding with oxygen residues via hydroxyls. Preparation and characterization of AA-rGO hybrids are described in detail in the ESI.† The resulting AA-rGO is readily water soluble, and is nonfluorescent upon excitation due to strong energy transfer between the fluorescence moiety anthracene and graphene. Upon exposure of AA-rGO to target molecules, AA molecules are released from the rGO and form an AA-target complex allowing the recovery of anthracene fluorescence. Therefore, the chiral sensing can be achieved through the enantioselective inclusion of amylose chains.^15–19

Scheme 1 Schematic representation of the regenerable chiral sensor using anthracene-labeled amylose-modified rGO (AA-rGO) hybrids based on the conformation alteration of AA for its release from graphene upon the chiroselective association to the guest molecules.

Materials and methods

Materials

G oxide was synthesized by a modified Hummers method^31,32 from graphite powder (Alfa Aesar, −20 + 80 mesh). Amylose (with an average degree of polymerization 100) was brought from Sinopharm Chemical Reagent Co. (Shanghai, China). 2-Aminoanthracene, D-tryptophan (Trp), L-Trp, D-tyrosine (Tyr), L-Tyr, and L-phenylalanine (Phe) were purchased from Sigma-Aldrich (Steinheim, Germany). Dialysis membrane was purchased from Pierce (3000 MWCO). All other regents were of analytical reagent grade. All aqueous solutions were prepared using ultra-pure water (18.2 MΩ, Milli-Q, Millipore).

Measurements

Fluorescence measurements were carried out using a JASCO FP-6500 spectrofluorometer with the slit width for the excitation and emission of 5 nm. Ultraviolet-visible spectroscopy (UV-vis) measurements were recorded on a Jasco-V550 UV-vis spectrophotometer. Fourier transform infrared spectroscopy (FTIR) measurements were carried out with a BRUKER Vertex 70 FTIR spectrometer. The sample was prepared as pellets using spectroscopic grade KBr. Circular dichroism (CD) spectra were measured on a JASCO J-810 spectropolarimeter equipped with a temperature controlled water bath. The optical chamber of the CD spectrometer was deoxygenated with dry purified nitrogen (99.99%) for 45 min before use and the nitrogen atmosphere was kept during the experiments. Three scans were accumulated and automatically averaged. The thermogravimetric analysis (TGA) was recorded with a RIGAKU Standard type with a heating rate of 10 °C min⁻¹ from room temperature to 800 °C. Atomic-force microscopy (AFM) measurements were performed using a Nanoscope V multimode atomic force microscope (Veeco Instruments, USA) under ambient conditions, and samples were prepared by dropping the solution on mica. Transmission electron microscopic (TEM) experiments were performed using a Philips Tacnai G2 20 S-TWIN microscope operating at 200 kV. For visualization by TEM, samples were prepared by dropping a solution of production on a copper grid. 1H nuclear magnetic resonance (NMR) spectra were recorded in a dimethyl sulfoxide (DMSO)-d₆ solution at 25 °C on a Bruker Avance 600 MHz NMR Spectrometer.

Preparation and characterization of AA-rGO hybrids

For the preparation of the AA-rGO hybrids, the homogeneous G oxide dispersion was mixed with AA aqueous solution and sodium hydroxide, and then the mixture was reduced with hydrazine monohydrate at 80 °C for 24 h.^30,32 After reduction, a black dispersion was obtained, and the excess of AA was removed with five successive cycles that involved centrifugation, decantation, and resolubilization to ultimately yield AA-rGO hybrids. The convincing characterizations of AA-rGO hybrids with UV-vis, FTIR, TEM, AFM and TGA are described in detail in the ESI.†

Preparation of anthracene-labeled amylose (AA)

This compound was synthesized by forming a Schiff base between the reducing end of amylose and –NH₂ of 2-aminoanthracene as described by Hanashiro and Takeda.²⁸ Amylose (200 mg) was dissolved in 30 mL DMSO/water (1 : 1, v/v), and a 1 mL 12 M HCl solution was added dropwise. After was added 2-aminoanthracene (20 mg), the system was stirred at 60 °C for 8 days. During the reaction, 20 mg sodium cyanoborohydride was added at intervals of 24 h. After the reaction completion, the reaction mixture was poured into 800 mL of ethanol to form a precipitate, which was washed with ethanol, and dried superficially. The dried precipitate was dissolved in 20 mL deionized water and the solution was further dialyzed (3000 MWCO) for a week to ensure complete removal of salts and excess 2-aminoanthracene. Ethanol was used to precipitate the amylose compounds from the dialyzed solution once more. The precipitate was then dried in a vacuum at room temperature for 48 h to give AA in 95% yield. ¹H NMR³³ (600 MHz, DMSO-d₆, ppm) δ 8.36, 7.94, 6.65, 6.25 (9H, H of anthracene), 5.06–5.09 (C-1 H of amylose), 3.30–3.64 (C-2, C-3, C-4, C-5, C-6 H of amylose), 5.46–5.48, 5.43–5.42, 5.57 (–OH of amylose).

Fluorescent chiral assays

In a typical chiral assay, a 10 μg mL⁻¹ AA-rGO solution was mixed with the target amino acid enantiomer in a 25 mM phosphate buffered saline (PBS). After 10 min incubation, fluorescence measurements were performed with a quartz cuvette to monitor fluorescence emission intensity within the range of 420–700 nm. All the measurements were performed at room temperature.

Results and discussion

As a proof of the concept, we first examined the G-based chiral sensor for the detection of amino acids, and Trp enantiomers. The aqueous solution of AA-rGO hybrids was directly used for sensing, and the AA concentration was calculated according to the TGA results (50 wt% of AA in AA-rGO hybrids, ESI†). Fig. 1 shows the fluorescence emission spectra of AA under different conditions. The fluorescence spectrum of AA in PBS in the absence of rGO showed that AA had strong fluorescence. However, for the AA-rGO hybrid, up to 98.5% fluorescence was quenched (Fig. 1, curve c). This observation indicated strong adsorption of AA on rGO and high fluorescence quenching efficiency of rGO. Intriguingly, upon addition of L-/D-Trp, the AA-G hybrids displayed chiral-selective fluorescence enhancement (Fig. 1, curves d and e). The fluorescence of the free AA was, however, scarcely influenced by the addition of L-Trp. The inset in Fig. 1 compares the fluorescence intensity ratio (F/F₀) of AA and AA-rGO upon addition of L-Trp, where F₀ and F are the fluorescence intensity at 500 nm in the absence or presence of L-Trp, respectively. In the case of AA alone, fluorescence was hardly changed even with a high concentration of L-Trp. However, for the AA-rGO hybrids, up to 70-fold fluorescence increase was observed. This high fluorescent enhancement made the sensing readily visible to the naked eye under a UV-lamp.

Fig. 1 Fluorescence emission spectra of AA (5 μg mL⁻¹) at different conditions: a) AA in PBS; b) AA + 500 μM L-Trp; c) AA-rGO; d) AA-rGO + 500 μM L-Trp; e) AA-rGO + 500 μM D-Trp. All the measurements were carried out in 25 mM PBS (pH 6). The AA-rGO concentration was kept at 10 μg mL⁻¹. Inset: fluorescence intensity ratio of AA-rGO and the control (AA) with 500 μM L-Trp. Excitation: 400 nm; emission: 500 nm.

It should be noted that the G-based sensor can be implemented for other target molecules when two criteria are fulfilled: (i) the analyzed target associates to AA by forming an inclusion complex that allows the release of AA from the rGO surface. (ii) the target does not quench the fluorescence of the labeled-dye. In the present study, the second criteria can be readily fulfilled for a wide range of targets due to the long excitation (>350 nm) and emission (>450 nm) wavelength. For verifying that the first criteria is the case for the G-based sensor comes into effect, the formation of an inclusion complex between AA and the target molecule needs to be proven in the presence of rGO.

The inclusion ability of G adsorbed AA was first proven by CD spectroscopy. Iodine was chosen as a model target because it is well-known that iodine can readily form helical complexes with amylose,³⁴ and produce evident CD bands.³⁵ The CD spectra of AA and AA-rGO hybrids with or without iodine were measured (Fig. 2A). No CD band was observed for both AA and AA-rGO themselves in the spectral region from 250 to 700 nm. In the presence of iodine, three iodine-amylose complex-characteristic CD bands were observed for AA solution, two positive bands near 350 and 550 nm, and a negative band near 700 nm.³⁵ The same CD bands with weaker intensity were observed for AA-rGO after adding iodine, indicating that the adsorbed AA can form an inclusion complex with iodine. The decreased CD intensity can be due to the inhibition effect of rGO. Moreover, similar results were observed when Trp enantiomers were used as target molecules. Both CD spectra of D-, L-Trp were changed upon addition of AA-G, suggesting inclusion association between the adsorbed AA and the D-, or L-Trp enantiomer (Fig. S1, ESI†).

Fig. 2 (A) CD spectra of 0.05 wt% AA, 0.1 wt% AA-rGO, 0.05 wt% AA+ 0.2 mM iodine, and 0.1 wt% AA-rGO + 0.2 mM iodine in 25 mM PBS (pH 6). (B) Fluorescence emission spectra (excitation at 400 nm) of AA-rGO hybrids in DMSO/PBS mixture containing DMSO 0–50 vol%. Other conditions were the same as those described in Fig. 1.

Before we could attribute the fluorescence recovery of the G-based sensor to the formation of inclusion complexes, further studies were carried out. First, the low dielectric constant (ε = 47.2) solvent DMSO is known to induce a conformational transition of amylose to helical structures by strengthening the inter residue hydrogen bonds between adjacent sugar rings of the amylose backbone.^36–39 Coincidentally, the fluorescence emission of AA-G hybrids was gradually enhanced with increasing DMSO (Fig. 2B). In addition, control experiments indicated that the fluorescence of AA alone was hardly changed with increasing DMSO (Fig. S2, ESI†). Thus, when DMSO was added to AA-rGO solutions, AA was induced to form the inclusion complex-like helical structures leading to the release of AA from rGO and recovery of the fluorescence. Second, since boric acid has been known to associate with cis-orientations of diols presented on polysaccharide backbones without forming inclusion complexes,^40–42 fluorescence emission of AA-rGO should not change in the presence of boric acid. As expected, fluorescence enhancement was not observed upon adding a high concentration (100 mM) of boric acid to AA-rGO (Fig. S3, ESI†), indicating that non-inclusion associations did not contribute to the fluorescence recovery. These results further support our hypothesis for the construction of a G-based chiral sensor. Fluorescence resonance energy transfer (FRET) experiments using 1-pyrene-methyl amino (PyMA) and Rhodamine 6G (R6G) indicate that FRET between AA-rGO and PyMA takes place because PyMA can be included inside the helical cavity of amylose (Fig. S4, ESI†). Since the structure and size of R6G determines that R6G cannot be included inside the helical cavity of amylose, no FRET between AA-rGO and R6G occurs (Fig. S5, ESI†). FRET results provide additional support that the fluorescence recovery of the G-based sensor is due to the helical complex formation.

Of particular importance for a chiral sensor is its chiral selectivity against the enantiomers. Higher chiral selectivity would allow wider practical applications. With this in mind, we compared F_L/F_D, the ratio of the fluorescence enhancement of G-based chiral sensor against D-/L-Trp. As shown in Fig. 3A, L-Trp binding resulted in significant fluorescence enhancement, whereas a much weaker fluorescence increase was observed for D-Trp at the same concentration of 75 μM. The chiral selectivity (F_L/F_D) of this G-based chiral sensor toward Trp enantiomers is up to 3.7, showing that the chiral selectivity of a G-based sensor is better than recently reported chiral sensors.^43–45 Besides, the chiral sensing and the concomitant fluorescence difference are visible under a UV lamp. In the presence of the same concentration of D- and L-Trp enantiomers (see inset of Fig. 3A), the color of the solutions are blue and green, respectively. A 14 nm bathochromic shift (from 489 to 503 nm) of the emission peak is responsible for the blue to green color change, and such an emission color difference can be readily distinguished by the naked eye. The color change could be attributed to the microenvironment-sensitive fluorescence property of the anthracene.^46,47 Time-dependent experiments (Fig. 3B) show that fluorescence enhancement of the G-based sensor against L-Trp takes place as a rapid enhancement in the first 2 min, then follows a slower increase over the next 6 min. This indicates that the reaction is complete within 10 min and the sensing is fast.

Fig. 3 (A) Fluorescence emission spectra in of AA-rGO in the absence and presence of 75 μM D-, or L-Trp. Inset: a photograph for AA-rGO with 75 μM D-, or L-Trp excited by a hand-held UV lamp. (B) Time-dependent spectral changes of the AA-rGO chiral sensing system: (a) In the absence of L-Trp, (b) to (f) upon addition of L-Trp 75 μM. Spectra recorded at time intervals of 2 min. Other conditions were the same as those described in Fig. 1.

Next control experiments were carried out to evaluate the selectivity of the G-based platform for Trp toward other essential amino acids since they may coexist with Trp in many samples (Fig. 4). The other two aromatic amino acids, Tyr and Phe, associate to AA much weaker than Trp, and do not interfere in the determination of Trp. Except for aromatic amino acids, very high concentrations (10 mM) of other amino acids, including cysteine (Cys), arginine (Arg), serine (Ser), threonine (Thr), isoleucine (Ile), and histidine (His), can only induce little fluorescence recovery of AA, indicating a strong anti-interference ability of the G-based sensor towards these nonaromatic amino acids.

Fig. 4 Selectivity of the G-based platform for L-, D-Trp toward other essential amino acids. L-Trp, D-Trp, L-Tyr, D-Tyr, and L-Phe were tested at 100 μM, and other nonaromatic amino acids were tested at 10 mM. Other conditions were the same as those described in Fig. 1.

Trp selectivity is from its specific interaction with amylose.^48–50 Previous studies have demonstrated that Trp residues situated at the active sites of glucoamylase are essential for inducing a conformational change in amylose.^48–50 When the Trp residues are mutated by other amino acids, the glucoamylase loses enzymatic activity towards its substrate amylose. The results further support our observed specific selectivity of the G-based platform for Trp towards other amino acids. As mentioned above, the different effects of Trp enantiomers and other amino acids on the fluorescence enhancement of the G-based sensor can be rationalized as the selective formation of inclusion complexes of AA. Competitive binding experiments were carried out to confirm whether amylose chains indeed associate to Trp with high selectivity and chiral preference. For direct visualization of amino acids binding, the amylose-iodine complex was used to observe the mixture color change in Eppendorf tubes (Fig. 5A). The amylose-iodine complex shows dark blue without adding amino acids (Fig. 5A, blank). Except L-/D-Trp, the other amino acids including L-/D-Tyr, L-Phe, L-Cys, L-Arg, L-Ser, L-Thr, L-Ile, and L-His, do not change the solution color. However, upon addition of 100 μM L- or D-Trp, the color changes from dark blue to faint-red and pale-blue, respectively. Unexpectedly, the simple amylose-iodine system can distinguish the subtle chiral difference of Trp enantiomers with a different color. To the best of our knowledge, this has not been reported before. For further study of the enantiodifference, we measured the time-dependent absorption change of the amylose-iodine in the presence of L- or D-Trp (Fig. 5B and C). After adding L- or D-Trp for 30 min, the absorption bands were shifted from approximately 600 to 540 and 571 nm, respectively. Previous studies have demonstrated⁵¹ that the color of the amylose-iodine complex is related to the length of the amylose chain. Red color (500–520 nm) indicates that the amylose-iodine complex is formed by amylose chains of 8 to 13 glucose units, purple color (520–540 nm) indicates the complex formed by chains of 13 to 40 units, and blue color (540–600 nm) indicates the complex formed by chains over 30 units. Thus, according to the color change⁵¹ and enzyme mutation studies,^48–50L-Trp can strongly bind to the long chain amylose-iodine complexes (blue) by competitively replacing the iodine out of the helical cavities and results in the breakdown of the long chain complex to the short segments (faint red).⁵¹

Fig. 5 (A) Photograph for 0.1 wt% amylose and 0.2 mM iodine in PBS (pH 6) without and with 100 μM aromatic amino acids or 10 mM nonaromatic amino acids, respectively. The tested other amino acids include L-Cys, L-Arg, L-Ser, L-Thr, L-Ile, and L-His. (B) and (C) Time-dependent visible absorption spectra of the amylose (0.1 wt%)-I₂ (0.2 mM) system with 100 μM L-Trp and D-Trp, respectively.

Another important practical challenge for the G-based chiral sensor is detection sensitivity. Fig. 6 showed the calibration curves of the G-based sensor in the presence of different concentrations of D-, or L-Trp. As the concentration of L-Trp increased, the fluorescence of the sensing system was significantly increased. However, D-Trp had less effect on the fluorescence enhancement. Favorable linear correlations (R² > 0.9) existed between the emission intensity at 500 nm and the concentration of D-, or L-Trp over the range of 0–5 μM (Fig. 6 inset). The limits of D-, L-Trp detection, based on 3σ/slope, were estimated to be about 134 nM and 34 nM, respectively. The detection sensitivity toward L-Trp is over 100-times higher than that of recently reported electrochemical sensors⁵² and colorimetric sensors.⁵³ This indicates that the designed G-based sensor has high chiroselectivity and sensitivity to Trp enantiomers.

Fig. 6 Calibration curves corresponding to the analysis of variable concentrations of D- and L-Trp by the G-based chiral sensor. The fluorescence intensity was recorded after equilibration of the system in the presence of different concentrations of Trp enantiomers for 10 min at 500 nm. Inset: linearity of fluorescence intensity against Trp enantiomers within the range of 0–5 μM. Other conditions were the same as those described in Fig. 1.

This G-based chiral sensor was then used to examine the enantiomer excess (ee) of Trp samples. The observed fluorescence intensity at 500 nm was gradually increased as the excess of L-Trp increased. Significantly, the plot of fluorescence against ee showed a linear dependence for Trp samples (Fig. S6, ESI†). The dependence allows the evaluation of the enantiomeric composition of Trp samples. It should be noted that the determination of the optical purity of Trp is important because of the difference in nutrition and biological functions of Trp enantiomers.^54,55

The regeneration is another important advantage of the designed G-based chiral sensor. To test this, AA-rGO hybrids were precipitated out of the sensing solution upon addition of ethanol and were readily collected by centrifugation (3300 rpm, 5 min) after the first sensing of L-Trp. The collected AA-rGO hybrids were then washed with hot PBS/EtOH (60 : 40, v/v; ca. 60 °C) 5 times. The PBS/EtOH was kept hot for the purpose of unwinding the inclusion complexes of AA and L-Trp,¹⁶ allowing the release of L-Trp and re-adsorption of AA onto the rGO. The washed AA-rGO hybrids were swept with nitrogen to remove EtOH and redispersed in PBS to the original volume for regeneration cycle. The AA-rGO hybrids were nonfluorescent after regeneration and a recovery of fluorescence was observed after adding L-Trp. As shown in Fig. 7, the AA-rGO hybrids can still effectively detect L-Trp after regenerated for three cycles. This demonstrates the reusability of this novel chiral sensor and its superiority in practical and economical applications relative to other G-based sensors.

Fig. 7 Test of regeneration of AA-rGO hybrids. The L-Trp was added to a concentration of 100 μM. Other conditions were the same as those described in Fig. 1.

Conclusions

In summary, we have designed for the first time a G-based chiral sensor. It is reusable and highly sensitive and selective. In comparison with commonly used sensors using DNA or peptide as probes, natural amylose is more attractive because of its low cost, ready availability, simple manipulation and regeneration. The specific selectivity for Trp enantiomers toward other essential amino acids allows potential chiroselective analysis of Trp in complex samples such as biological fluids. This design can, in principle, be implemented for other slender target molecules that can form inclusion complexes with amylose.

Acknowledgements

This work was supported by 973 Project (2011CB936004), NSFC (20831003, 90813001, 20833006, 90913007) and Funds from the Chinese Academy of Sciences.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c1sc00308a

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