A FRET-based fluorescent approach for labetalol sensing using calix[6]arene functionalized MnO₂@graphene as a receptor

Abstract

A turn-on fluorescent sensing platform for labetalol determination has been developed based on competitive host–guest interaction between p-sulfonated calix[6]arene (SCX6) and signal probe/target molecules by using SCX6 functionalized MnO₂@reduced graphene oxide (SCX6-MnO₂@RGO) as a receptor. Rhodamine 6G (R6G) and labetalol were selected as the probe and target molecules, respectively. When R6G enters into the SCX6 host, its fluorescence is quenched by MnO₂@RGO. However, on addition of labetalol to the preformed R6G·SCX6-MnO₂@RGO complex, the R6G molecule is displaced by labetalol from the host of SCX6, leading to a “switch-on” fluorescence response. This is due to the fact that the binding constant of the labetalol/SCX6 complex is much higher than that of R6G/SCX6. The fluorescence intensity of the SCX6-MnO₂@RGO·R6G complex increased linearly with increasing concentration of labetalol ranging from 1.0 to 18.0 μM. The proposed method showed a detection limit of 0.25 μM for labetalol. In addition, 2D NMR and molecular modeling studies indicated that the salicylamide part of the labetalol molecule inserted into the cavity of SCX6, while the phenylpropyl group located outside of the SCX6 host.

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1. Introduction

Fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer from a donor to an energy acceptor at close distances (i.e. 1–10 nm). FRET has been widely utilized in the fluorescent sensing field due to its advantages of high sensitivity and suitability for homogeneous detection.¹ In a FRET system, the energy transfer efficiency is an important parameter for assay sensitivity, which greatly depends on the emission properties of the donor and the absorption capability of the acceptor.² Graphene has attracted considerable attention because of the unique thermal, mechanical, and electronic properties arising from its strictly 2D structure, and to its potential technical applications.^3,4 Graphene has also been increasingly explored as a new excellent quencher for fluorescent probes in chemical and biological fields due to its remarkable high fluorescence quenching efficiency.⁵ Layered transition-metal dioxides or disulphides (LTMDs) such as MoS₂, WS₂, and MnO₂ are a class of 2D nanomaterials that have been explored for widespread applications in energy generation,⁶ sensing,⁷ photocatalysis,⁸ and photothermal therapy.⁹ In addition, LTMDs have been demonstrated as promising fluorescence quenchers and attracted increasing attention in constructing FRET-based bio/chemical sensing platforms due to their excellent light absorption capability and fast electron transfer rate.¹ Recently, a lot of FRET-based fluorescent sensing platforms have been developed by many research groups using the 2D graphene or LTMDs as energy acceptors.^10–18 In these studies, a dye-labeled aptamer or ssDNA was usually designed as energy donors, which self-assembled with 2D nanosheets via π–π stacking to form nanoprobes and were applied for the detection of biomolecules. The aforementioned method is effective and feasible to obtain a “turn on” fluorescent sensing platform. Nonetheless, such surface modification and biological labeling are obligatorily needed, usually resulting in time-consuming and complicated procedures to some degree.^1,5 Therefore, it is still highly desirable to develop a simple and convenient approach to gain a “turn on” fluorescent sensing platform.

In recent years, researchers are pushing ahead to develop the nanohybrids of macrocyclic hosts (e.g. cyclodextrins, calixarenes) and carbon materials (e.g. CNT, CNH, graphene) for various technological applications such as electrochemical/optical sensing, bioimaging, drug delivery, and so on. Calixarenes, recognized as the third class of macrocyclic host after crown ethers and cyclodextrins, which show high supramolecular recognition with various guest molecules.^19,20 Especially, the p-sulfonated calixarenes have been widely investigated for various applications due to their high water-solubility. It has been reported that the composites of calixarenes and carbon materials could be formed by π–π interactions and hydrogen interactions.^21–23 If MnO₂@graphene are modified with water-soluble calixarenes, it is possible to obtain new functionalized materials that simultaneously possess the unique properties of MnO₂@graphene (excellent quenching performance) and calixarenes (high supramolecular recognition capability) through combining their individual characteristics. More importantly, a facile competitive fluorescent sensing platform could be obtained by using such a calixarene–MnO₂@graphene composite as receptor. Compared with the surface modification approach, such a competitive fluorescent sensing platform decreased the difficulty of synthesis and expanded the application of these 2D materials since it does not need the biological labeling process. Thus, such calixarene–MnO₂@graphene composite could potentially applied in the fluorescent sensing field to arouse extensive research interest.

Labetalol hydrochloride, as a noncardiovascular β-blocker, reported to possess some intrinsic sympathomimetic and membrane stabilizing activity, can reduce heart rate and tremor.²⁴ Thus, it has been added to the list of forbidden substances issued by the International Olympic Committee.²⁴ In the present work, labetalol was selected as a model analyte, by combing the merits of p-sulfonated calix[6]arene (SCX6) and MnO₂@reduced graphene oxide (MnO₂@RGO), a facile FRET-based turn-on fluorescent approach for labetalol sensing based on a competitive host–guest recognition between SCX6 and signal probe/target molecules was developed by using SCX6 functionalized MnO₂@RGO (SCX6-MnO₂@RGO) as a receptor. The design principle of the developed fluorescent sensing platform for labetalol sensing is illustrated in Fig. 1. Rhodamine 6G (R6G) and labetalol were selected as the probe and target molecules, respectively. When R6G enters into the SCX6 host, its fluorescence is quenched by MnO₂@RGO. However, upon the presence of labetalol to the performed R6G·SCX6-MnO₂@RGO complex, the R6G molecules are displaced by labetalol from the host of SCX6, leading to a “switch-on” fluorescence response.

Fig. 1 Fluorescent approach for labetalol sensing using R6G·SCX6-MnO₂@RGO as receptor.

2. Materials and methods

2.1. Chemicals

Graphite oxide was purchased from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). p-Sulfonated calix[6]arene hydrate (SCX6, Fig. S1†) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). R6G and labetalol (Fig. S2†) were provided by Shanghai Adamas Reagent Co., Ltd. (Shanghai, China). Human serum sample was purchased from Biocell Co. (Zhengzhou, China). All aqueous solutions were prepared with deionized water (DW, 18 MΩ cm). All other reagents were of analytical grade.

2.2. Apparatus

The morphology of the obtained samples were characterized by a JEM 2100 transmission electron microscopy (TEM, Japan) equipped with an energy dispersive X-ray spectrometry (EDX). Fourier transform infrared (FTIR) study was performed over the wavenumber, range of 4000–400 cm⁻¹ by a Thermo Fisher SCIENTIFIC Nicolet IS10 (Massachusetts, USA) FTIR impact 410 spectrophotometer using KBr pellets. Raman spectra were obtained on a 400F PERKIN-ELMER Raman spectrometer (Shelton, USA) with a 514.5 nm wavelength incident laser light. Thermogravimetric analysis (TGA) was carried out on a Q50 TGA (TA Instruments, New Castle, USA), from 25 to 800 °C in argon at a heating rate of 5 °C min⁻¹. X-ray photoelectron spectroscopy (XPS) measurements were performed with Al Ka X-ray radiation as the X-ray source for excitation, which were carried out on an ESCALAB-MKII spectrometer (VG Co., United Kingdom). A Malvern Zetasizer Nano series was used for the zeta potential measurements. Fluorescence titration experiments were carried out using a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan) at room temperature. The 2D NMR spectra were recorded on a Bruker AV. DRX5 instrument operated at 500 MHz.

2.3. Stoichiometry determination

The stoichiometry of the SCX6/labetalol complex was determined using the continuous variation of Job's method by fluorescence spectroscopy.²⁵ The total molar concentration of the guest and host aqueous mixture was kept constant at 50 μM. The fluorescence was recorded at the different molar ratios varying from 0 to 1.

2.4. Preparation of the SCX6/labetalol inclusion complex

The inclusion complex of SCX6/labetalol was prepared via the freeze-drying method. A 1 : 1 molar ratio of labetalol and the SCX6 were weighted accurately and dissolved in DW. The labetalol aqueous solution was added slowly into the solution of SCX6, and the mixed solution was then stirred at room temperature for 24 h. The resulting suspension was filtered through a 0.22 μm filter membrane. The clear solution was frozen, and then lyophilized to obtain the SCX6/labetalol inclusion complex.

2.5. Molecular docking

The crystal structure of “winged cone” conformation of SCX6 (ID: FEQYOQ) were obtained from Cambridge Crystallographic Data Centre (CCDC). The structures of “pinched cone” conformation²⁶ of SCX6 and of labetalol were constructed by the UCSF Chimera software. Both the “winged cone” SCX6, “pinched cone” SCX6 and labetalol structures were fully optimized by the UCSF Chimera software. Hydrogen atoms were added using the Dock Prep module. The GAFF force field and the AM1-BCC charges were used. The optimized structures were used as a starting structure in the docking study. For the molecular docking study, the molecular surface of the SCX6 molecule was generated using the DMS tool with a probe radius of 1.4 Å. The sphgen module was applied to generate spheres surrounding the binding site. The grid module of DOCK6 was employed to generate grid file which was used in scoring in the subsequent docking procedure. The flexible docking method was utilized producing 1000 different conformational orientations for the guest molecule. The electrostatic interactions and van der Waals forces were calculated based on the grids. Finally, clustering analysis with root-mean-square deviation (RMSD) threshold of 2.0 Å was performed to retain the best results.

2.6. Preparation of the SCX6-MnO₂@RGO composite

Graphite oxide was exfoliated into graphene oxide (GO) sheets by ultrasonication at room temperature for 1 h. A green wet-chemical approach³ reported by Zhang' group with some modifications was used for the preparation of the SCX6-MnO₂@RGO composite. In a typical synthesis, 50 mL of mixture aqueous solution containing 0.01 M Na₂SO₄ and 0.001 M KMnO₄ was added into 10 mL of 1.0 mg mL⁻¹ GO suspension. After the pH was adjusted to 12.0 using 1.0 M NaOH, the mixture was transferred to a round bottom flask and stirred at 80 °C in an oil bath for 8 h. Then 20.0 mg SCX6 was added into the above mixture and continuous stirred at 80 °C for 8 h. After cooling to room temperature, the resulting stable black dispersion was centrifuged at 16 000 rpm and washed with DW for three times. Finally, the resulting SCX6-MnO₂@RGO was obtained by freeze-drying.

2.7. Fluorescence titration experiments

Stock solutions of R6G (400 μM), labetalol (400 μM), and SCX6-MnO₂@RGO (1.0 mg mL⁻¹) in DW were prepared. The dye stock solution was diluted with DW to a final concentration of 10 μM. The SCX6-MnO₂@RGO was then gradually added until the dye fluorescence was quenched. For the competitive displacement of dye from the SCX6-MnO₂@RGO by labetalol, the required amount of labetalol was gradually added into the performed R6G·SCX6-MnO₂@RGO complex. The combined solution was mixed by vortexing well for 5 min before the fluorescence was recorded.

2.8. Determination of labetalol in human serum samples

The determination of labetalol in human serum was performed. The serum sample was diluted 50 times. A stock solution of 500 μM labetalol was prepared. Then the known amounts of labetalol were added into the serum sample. Finally, this solution was used to detect labetalol according to the procedure described above.

3. Results and discussion

3.1. Characterization of the SCX6-MnO₂@RGO composite

The obtained SCX6-MnO₂@RGO could be stably dispersed in water even after removing free SCX6 via high-speed centrifugation (16 000 rpm) and no obvious precipitates are observed after being stored for more than 6 months (Fig. S3†). The zeta potential measurements of the MnO₂@RGO and SCX6-MnO₂@RGO were obtained as shown in Fig. 2. The average zeta potentials of MnO₂@RGO and SCX6-MnO₂@RGO were −36.1 and −39.3 mV, respectively. The zeta potential of SCX6-MnO₂@RGO decreased approximately 3 mV compared with that of MnO₂@RGO, which was ascribed to the negative charge of –SO₃⁻. Generally, all the zeta potentials were lower than −30 mV, suggesting that the colloidal stability of the MnO₂@RGO and SCX6-MnO₂@RGO dispersions was very high. The TEM characterizations of RGO and MnO₂@RGO were obtained. The RGO consists of randomly aggregated thin, wrinkled sheets closely associated with each other (Fig. 3A). As shown in Fig. 3B, the most striking feature is that the MnO₂ with a uniform size about 10 nm are fairly well dispersed on the surface of RGO, indicating the formation of the MnO₂@RGO composite. As shown in Fig. 3C, the FTIR spectrum of RGO displays the stretching vibrations of –OH (3440 cm⁻¹), C–O (1200 cm⁻¹), and C–O/C–C (1090 cm⁻¹) due to a small amount of the remaining oxygen-containing functional groups.²⁷ The C=C (1630 cm⁻¹) conjugation is observed in RGO.²⁸ The FTIR spectrum of MnO₂@RGO was similar to that of RGO. In the case of SCX6-MnO₂@RGO, the peaks of –OH (3440 cm⁻¹) and O–H bending vibrations (1401 cm⁻¹) enhanced obviously, which may be caused by the introduced –OH in SCX6. The Raman spectra of RGO, MnO₂@RGO, and SCX6-MnO₂@RGO samples are illustrated in Fig. 3D. The characteristic D and G bands of RGO are observed at 1355 and 1600 cm⁻¹, respectively.²⁹ The MnO₂@RGO shows higher intensity ratio of D band and G band (I_D/I_G) (0.86) than the RGO (0.84), suggesting a decrease in sp² domains and a higher concentration of structure defects on the MnO₂@RGO.³⁰ The Raman spectrum of SCX6-MnO₂@RGO was similar to that of MnO₂@RGO, indicating that the structure of MnO₂@RGO is not affected by SCX6.³¹ The EDX analysis of MnO₂@RGO was illustrated in Fig. 3E. It can be clearly noticed that the Mn, C, and O elements were presented in the MnO₂@RGO composite, suggesting that the composite is made up of MnO₂ and RGO. On the basis of the quantitative analysis of the EDX data, the atomic percentages of C, O, and Mn in the composite are estimated to be 49.12, 41.02, and 9.86%, respectively, from which the weight ratio of the MnO₂ component in MnO₂@RGO is estimated to be approximately 47.94 wt%. The prepared SCX6-MnO₂@RGO was characterized by TGA, as shown in Fig. 3F. For the MnO₂@RGO, the minor loss in mass (9 wt%) at a temperature of approximately 200 °C was due to the pyrolysis of a very small amount of the remaining oxygen-containing functional groups in RGO. The mass loss of the SCX6-MnO₂@RGO reached about 41 wt% when the temperature was 200 °C. To deduct the mass loss of MnO₂@RGO, the mass loss caused by the decomposition of SCX6 was 32 wt%. Therefore, all these results demonstrated that the SCX6 had successfully grafted on the MnO₂@RGO.

Fig. 2 Zeta potentials of MnO₂@RGO (A) and SCX6-MnO₂@RGO (B).

Fig. 3 TEM images of RGO (A) and MnO₂@RGO (B); FTIR spectra of RGO, MnO₂@RGO, and SCX6-MnO₂@RGO (C); Raman spectra of RGO, MnO₂@RGO, and SCX6-MnO₂@RGO (D); EDX analysis of MnO₂@RGO (E); TGA curves of MnO₂@RGO and SCX6-MnO₂@RGO (F).

XPS analysis was used to determine the electronic structure and compositions of MnO₂@RGO. As shown in Fig. 4A, the survey spectrum confirms the presence of Mn, C, and O in the composite. This result is in agreement with EDX result. The Na signal is attributed to residual NaOH. The high-resolution spectrum of Mn 2p in Fig. 4B obviously shows that the peaks centered at 642.0 and 653.7 eV with a spin-energy separation of ∼11.7 eV can be assigned to Mn 2p_3/2 and Mn 2p_1/2 peaks, respectively, confirming the presence of MnO₂ in the composites.^32–34 Fig. 4C is the C 1s spectrum, which reveals that there are three components of carbon bond, namely sp² C (284.6 eV), sp³ C (285.6 eV), C–O (286.7 eV), C=O (287.8 eV), and O–C=O (288.8 eV) are presented in the composites.^23,35 In addition, the spectrum of O 1s is depicted in Fig. 4D. The deconvolution peaks of O 1s spectrum can be divided into two peaks centered at 530.0 and 532.0 eV, which corresponds to O–Mn and O–C bonding configurations, respectively.^32,33 Based on the above analysis, the composite is made up of MnO₂ and RGO.

Fig. 4 XPS survey spectrum of MnO₂@RGO (A); the narrow spectra of Mn 2p (B), C 1s (C), and O 1s (D).

On the basis of the above experimental results, it could be concluded that the SCX6-MnO₂@RGO composite was successfully fabricated through a one-step wet-chemical method. The KMnO₄ can react with graphene to form MnO₂ at a moderate temperature of 80 °C.^33,36–38 According to equation: 4KMnO₄ + 3C + H₂O → 4MnO₂ + K₂CO₃ + 2KHCO₃, the MnO₂ could be anchored on RGO. The composites of calixarenes and carbon materials could be formed by π–π interactions, hydrogen interactions, and electrostatic interaction.^{21,23,39–41}

3.2. Stoichiometry of SCX6/labetalol

The stoichiometry for the inclusion complexation of SCX6 with guest labetalol were determined by Job's experiments by fluorescence spectroscopy. As shown in Fig. S4,† the plot maximum point appears at a SCX6 molar fraction of 0.5, which obviously indicates that a 1 : 1 inclusion complex is formed between SCX6 and labetalol.

3.3. The 2D ¹H NMR analysis of the labetalol/SCX6 complex

It is well known that the size/shape-matching, and induced-fit interaction occur in the molecular binding process of macrocyclic hosts, and therefore, it is very important to investigate the interaction binding models between host and guest molecules for elucidating the mechanism of molecular recognition. According to the relative intensity of the NOE cross-peaks, one may estimate the binding mode between the macrocyclic hosts and guest molecules.⁴² The 2D NMR spectroscopy has become an essential method for the study of the conformations of macrocyclic hosts and their complexes,⁴² since it may be concluded that two protons are closely located in space 0.4 nm apart at most if a clear Nuclear Overhauser Effect (NOE) cross-peak is detectable between the relevant protons in the NOESY or ROESY spectrum. On this basis, ROESY experiments were employed to investigate the inclusion geometry of SCX6 with labetalol in D₂O at 25 °C. Compared with the ROESY spectrum of labetalol (Fig. 5A), some new peaks appeared in labetalol/SCX6. The ROESY spectrum of an equimolar mixture of SCX6 with labetalol in D₂O was obtained as shown in Fig. 5B, which clearly displays NOE cross-peaks (peak A) between the H_a protons of labetalol and the aromatic protons H1 of SCX6, as well as between H_b/H_c of labetalol and H1 of SCX6 (peaks B and D). Meanwhile, the NOE cross-peaks (peak C) between H_a protons of labetalol and the protons H2 of SCX6 are observed, suggesting that the salicylamide part of the labetalol molecule was included into the SCX6 cavity.

Fig. 5 ¹H ROESY spectra of labetalol (A) and labetalol/SCX6 (B) in D₂O at 25 °C.

3.4. Molecular docking

Molecular docking was performed to study the SCX6/labetalol inclusion complex in order to gain an insight into the binding modes. The binding models of labetalol with SCX6 were simulated using the DOCK6 program. Initially, the docking scores of SCX6/labetalol complex were obtained and provided in Table 1. The lowest energy score of the “pinched cone” conformation complex (−45.35 kcal mol⁻¹) is slightly higher than that of the “winged cone” conformation (−44.11 kcal mol⁻¹). Moreover, the two conformation complex was optimized by the sander procedure of AmberTools15 and the binding free energy (ΔG_bind) was calculated by MM-GBSA procedure. As shown in Table 2, the ΔG_bind of the “pinched cone” and “winged cone” complexes calculated by MM-GBSA method (ΔG_bind = ΔG_vdw + ΔG_es + ΔG_pol + ΔG_apol) were −25.12 and −28.82 kcal mol⁻¹, respectively. The ΔG_bind of the “pinched cone” complex is a little less than that of “winged cone” complex. As shown in Fig. 6A and B, the lowest energy docked conformations of the “winged cone” and “pinched cone” complexes for SCX6/labetalol were obtained, respectively. The salicylamide part of the labetalol molecule was included into the cavity of SCX6 both in the two conformations, while the phenylpropyl group located outside of the SCX6 host. This result is in accordance with the 2D NMR results. For the “winged cone” conformation, it was stabilized by four hydrogen bonds, one between the O17 atom of SCX6 and the guest N atom (2.8 Å), one between the O3 atom of SCX6 and the guest O1 atom (2.8 Å), one between the O22 atom of SCX6 and the guest N1 atom (2.8 Å), and one between the O6 atom of SCX6 and the guest hydroxyl O atom of salicylamide (2.6 Å). Secondly, the salicylamide benzene ring of labetalol formed π–π interactions with one benzene ring of SCX6. Thus, the electrostatic force contribution provided by hydrogen bonding and π–π interaction was obtained as −51.62 kcal mol⁻¹. In addition, hydrophobic interactions could be observed between the nonpolar moiety of labetalol and benzene rings of SCX6. The corresponding van der Waals contribution was −27.37 kcal mol⁻¹. In the case of the “pinched cone” conformation, it was stabilized by three hydrogen bonds, one between the O14 atom of SCX6 and the guest N1 atom (3.1 Å), one between the O2 atom of SCX6 and the guest O1 atom (2.7 Å), and one between the O2 atom of SCX6 and the guest N atom (2.9 Å). The corresponding electrostatic interaction contribution was −53.40 kcal mol⁻¹. On the other hand, the van der Waals contribution provided by hydrophobic interactions was −28.12 kcal mol⁻¹. Overall consideration the results, we find that the ΔG_bind of the “winged cone” conformation of the SCX6/labetalol complex is approximately equal to that of the “pinched cone” conformation. The electrostatic interaction contribution and the van der Waals contribution is also nearly equal. Besides, it has been reported that the size or/and shape of the guest has the capacity to influence the shape of the calixarenes. The host–guest complexes between calixarenes and guests can mutually induced fit with each other, forming adaptive supermolecular systems due to the high conformational flexibility of the calixarenes.⁴³ Thus, we believe that the conformation of SCX6 would change after binding with the guest and the complex is in a dynamic balance of the two conformations.

Table 1 Molecular docking scores for SCX6/labetalol complex^a

Host	Guest	Grid_score (kcal mol^−1)	Grid_vdw (kcal mol^−1)	Grid_es (kcal mol^−1)	Int_en (kcal mol^−1)
a vdw: van der Waals force; es: electrostatic force; Int_en: intramolecular energy.
SCX6-winged	Labetalol	−44.111374	−40.912689	−3.198683	5.171838
SCX6-pinched		−45.34903	−33.419674	−11.929354	13.125495

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Table 2 The binding free energy was computed by MM-GBSA method^a

Host	Guest	ΔGvdw	ΔGele	ΔGpol	ΔGapol	ΔGbind
a ΔGvdw: van der Waals contribution; ΔGes: electrostatic contribution; ΔGpol: polar solvation contribution; ΔGapol: nonpolar solvation contribution; ΔGbind: binding free energy; ΔGbind = ΔGvdw + ΔGes + ΔGpol + ΔGapol.
SCX6-winged	Labetalol	−27.3681	−51.6192	53.5250	−3.3585	−28.8208
SCX6-pinched		−28.1196	−53.3966	59.4985	−3.0981	−25.1158

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Fig. 6 The typical conformation of the “winged cone” labetalol/SCX6 (A) and “pinched cone” labetalol/SCX6 (B) complexes by molecular docking.

3.5. Fluorescence spectra analysis

The fluorescence quenching performance of the SCX6-MnO₂@RGO and the related materials towards R6G was investigated. As shown in Fig. S5,† the fluorescence spectra of 10 μM R6G in the absence and presence of 1 μg mL⁻¹ MnO₂, RGO, MnO₂@RGO, and SCX6-MnO₂@RGO were obtained. The fluorescence quenching of MnO₂@RGO is significantly higher than that of MnO₂ or RGO, which was ascribed to the synergistic effects. Compared with MnO₂@RGO, the fluorescence quenching of SCX6-MnO₂@RGO enhanced further, indicating that the dye indicator inserted into the cavity of SCX6. Fig. 7A shows that fluorescence emission intensity of R6G progressively decreased with the increase of SCX6-MnO₂@RGO. To confirm that the main factor causing fluorescence quenching was MnO₂@RGO, control experiments were carried out. It is clear from Fig. 7C that the fluorescence intensity of R6G was quenched by approximately 60% in the presence of SCX6-MnO₂@RGO, while addition of free SCX6 caused a fluorescence quenching of approximately 10%, indicating that the fluorescence quenching of the dye is predominantly because of the energy transfer between the dye and MnO₂@RGO.^1,5 In contrast, as illustrated in Fig. 7B, the successive addition of labetalol to the performed R6G·SCX6-MnO₂@RGO complex led to a successive reversion of the fluorescence of R6G. The increased fluorescence signal is directly related to the amount of labetalol added, which suggested a fluorescence approach for the detection of labetalol. Control experiments have been performed in order to confirm that the observed fluorescence recovery is caused by the displacement of R6G by labetalol from the SCX6 host. As depicted in Fig. S6,† although the fluorescence quenching effect is significant for MnO₂@RGO, the quenched fluorescence is not “turned on” upon the addition of labetalol. Thus, it can be concluded that the dye indicator first formed inclusion complex with R6G·SCX6-MnO₂@RGO and then released from R6G·SCX6-MnO₂@RGO upon the addition of labetalol accompanied with a phenomenon of fluorescence “switch-off–on”. The calibration curves for labetalol quantification were obtained and depicted in Fig. 7D. The fluorescence ratio F/F₀ were proportional to the labetalol concentrations. The linear response ranges of 1.0–12.0 and 12.0–18.0 μM with a detection limit of 0.25 μM (S/N = 3) was obtained. The corresponding regression equations were calculated as F/F₀ = 0.069C (μM) + 1.12 and F/F₀ = 0.017C (μM) + 1.72. In addition, the quantitation parameters of the present method were compared with those of other reported methods for the determination of labetalol (Table 3). Compared with these reported approaches for labetalol detection, the present competitive fluorescent method is exhibited a lower detection limit and wider linear range and it is important that the present method is very simple and convenient.

Fig. 7 (A) The effect of increasing concentrations of SCX6-MnO₂@RGO on the fluorescence intensity of R6G (λ_ex = 490 nm). ST concentration was 10 μM. (B) Fluorescence spectra of the R6G·SCX6-MnO₂@RGO complex via different concentrations of labetalol. The combined solution was mixed by vortexing well for 5 min and then tested. (C) Fluorescence spectra of 10 μM R6G in the absence and presence of 1 μg mL⁻¹ SCX6 and SCX6-MnO₂@RGO. (D) Calibration curves of fluorescence intensity of R6G·SCX6-MnO₂@RGO vs. labetalol concentration.

Table 3 Comparison of some methods used for determination of labetalol

Methods	Linear range (μM)	Detection limit (μM)	Ref.
LC-MS-MS	—	0.55	44
CE	63.6–318.0	0.33	45
Spectrofluorimetry	2.74–41.1	2.16	46
Spectrophotometry	2.74–27.4	2.14	47
Spectrophotometry	0.034–1.23	0.012	24
Competitive fluorescence	1.00–18.00	0.25	This work

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3.6. The mechanism of the competitive host–guest interaction

In order to clarify the mechanism of the competitive host–guest interaction, the plots of fluorescence intensity of 10 μM R6G (Fig. 8A) and 10 μM labetalol (Fig. 8C) vs. SCX6 concentration and the double reciprocal plots of 1/(F₀ − F) versus 1/[SCX6] for R6G (Fig. 8B) and labetalol (Fig. 8D) to SCX6 were obtained, indicating the existence of the 1 : 1 complex.²² From the plots the binding constants (K) for the 1 : 1 R6G/SCX6 and labetalol/SCX6 complexes were calculated to be 3.10 × 10⁵ M⁻¹ and 1.58 × 10⁶ M⁻¹, respectively. The K value of labetalol/SCX6 complex was more than 5 times higher than that of R6G/SCX6, which demonstrated the stronger binding of labetalol with SCX6 than that with R6G.

Fig. 8 Fluorescence intensity of 10 μM R6G (A) and 10 μM labetalol (C) vs. SCX6 concentration and plots of 1/(F − F₀) vs. 1/[SCX6] for R6G (B) and labetalol (D). (λ_ex = 490).

3.7. Selectivity and analytical application

The selectivity for the determination of labetalol was studied with 5-fold concentration of two other analogues (atenolol and metoprolol). Besides, 100-fold concentration of interferences from common molecules such as ascorbic acid (AA), MgCl₂, KCl, NaCl, glucose, sucrose, SO₄²⁻, and NO₃⁻ were also tested. The changes in the fluorescence ratio (F − F₀)/F₀ of the R6G·SCX6-MnO₂@RGO complex upon addition of a competitive binding analyte were displayed in Fig. 9. Upon interaction with the competitive binding analytes, the fluorescence of R6G·SCX6-MnO₂@RGO was increased selectively by addition of labetalol, while addition of other competitive binding analytes caused nonsignificant fluorescence changes. The proposed method was also applied to the determination of labetalol using standard addition methods in human serum samples. The results were listed in Table 4. The recoveries were in the range of 97.8–104.0% and RSDs were in the range of 2.6–3.2%. As can be seen, the precision and accuracy of the proposed method were satisfactory, indicating that this method could be extended for detection of labetalol in human blood.

Fig. 9 Relative fluorescence intensity is calculated by (F − F₀)/F₀, where F₀ and F are the fluorescence intensity without and with the presence of 10 μM labetalol (a), 50 μM metoprolol (b), 50 μM atenolol (c), 1.0 mM AA (d), 1.0 mM MgCl₂ (e), 1.0 mM KCl (f), 1.0 mM NaCl (g), 1.0 mM glucose (h), 1.0 mM sucrose (i), 1.0 mM SO₄²⁻ (j), and 1.0 mM NO₃⁻ (k), respectively.

Table 4 Determination of labetalol in human serum samples (n = 6)

Sample	Added (μM)	Founded (μM)	RSD (%)	Recovery (%)
1	0	0.00	—	—
2	2	2.07 ± 0.18	3.1	103.5
3	4	4.16 ± 0.12	2.6	104.0
4	6	5.87 ± 0.19	3.2	97.8

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4. Conclusions

In summary, a facile turn-on fluorescent sensing platform using dye R6G and SCX6-MnO₂@RGO as the energy donor–acceptor pair is designed and fabricated. Due to the outstanding quenching performance of the MnO₂@RGO and the excellent host–guest recognition of SCX6, this sensing system demonstrates a practical application for the selective and sensitive detection of labetalol in human serum samples. This study reveals that the macrocyclic hosts–LTMDs@graphene composite would be superior energy acceptors for fabricating FRET-based assays. In addition, the inclusion complex of the SCX6/labetalol was studied by the molecular docking studies, which indicated that the salicylamide part of the labetalol molecule was included into the cavity of SCX6, while the phenylpropyl group located outside of the SCX6 host. The binding mode analysis demonstrated that the hydrogen bonding interactions, π–π interaction, and hydrophobic interaction contributed to form the inclusion complex.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21565029) and the Program for Excellent Young Talents, Yunnan University.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra14835b

‡ These authors contributed equally to this work.

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