Fluorescent Detection of Tadalafil Based on Competitive HostâGuest

Research Article www.acsami.org

Fluorescent Detection of Tadalafil Based on Competitive Host−Guest Interaction Using p‑Sulfonated Calix[6]arene Functionalized Graphene Long Yang,†,§ Hui Zhao,‡,§ Yucong Li,† Xin Ran,† Guogang Deng,† Xiaoguang Xie,† and Can-Peng Li*,† †

School of Chemical Science and Engineering, Yunnan University, Kunming 650091, People’s Republic of China Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: A competitive fluorescence method toward tadalafil detection has been developed based on host−guest recognition by selecting rhodamine B (RhB) and p-sulfonated calix[6]arene functionalized graphene (CX6−Gra) as the “reporter pair”. Upon the presence of tadalafil to the performed CX6−Gra−RhB complex, the RhB molecules are displaced by tadalafil, leading to a “switch−on” fluorescence signal. The observed fluorescence signal can be used for quantitative detection of tadalafil ranging from 1.00 to 50.00 μM with a detection limit of 0.32 μM (S/N = 3). The inclusion complex of tadalafil and CX6 was studied by molecular docking and the results indicated that a 1:1 host−guest stoichiometry had the lowest ΔG value of −7.18 kcal/mol. The docking studies demonstrated that the main forces including π−π interactions, electrostatic interactions, and hydrophobic interactions should be responsible for the formation of this inclusion compound. The mechanism of the competitive host−guest interaction was clarified. The binding constant (K) of the tadalafil/CX6 complex was more than 5 times greater than that of RhB/CX6. KEYWORDS: competitive host−guest interaction, calix[6]arene, graphene, fluorescence, tadalafil

■

grafted to the receptor, also constructed a “turn−off−on” Gra molecular sensing platform, which greatly reduced the synthetic difficulty and broadened its application in fluorescent sensing fields.6,8−10 Calixarenes, recognized as the third class of macrocyclic host molecules after crown ethers and cyclodextrins, have become important receptors because they can form stable host−guest complexes with various guest molecules, showing high supramolecular recognition and enrichment capability.11,12 It has been reported that the composites of calixarenes and carbon materials (e.g., carbon nanotube, Gra) could be formed by π−π interactions and hydrogen bonding interactions.13−16 The advantage of water-soluble calixarenes, particularly, psulfonated derivatives, as well as cyclodextrins functionalization is that it offers high water solubility to Gra and guest molecules incorporated into calixarenes are easily accessible to Gra.13,14 Tadalafil is one of the three selective phosphodiesterase type five inhibitors. It is the active constituent of Cialis, which is a prescription medicine approved by the United States Food and Drug Administration in 2003 for the therapy of erectile dysfunction.17 Because of its harmful side effects, the use of Cialis is controlled by medical supervision.18 Numerous analytical techniques including, HPLC with fluorescence,19 GC−MS,20 immunoassay,21 and electrochemical method22

INTRODUCTION In 1960, the first competitive binding assay for measuring plasma insulin was proposed by Berson and Yalow.1 After that, extensive applications based on competitive assays have received great attention.2 Along with the host−guest chemistry development, the conceptually so-called indicator displacement assays (IDAs) have attracted much attention to find out the potential of artificial receptors, especially macrocyclic host molecules, for its prospective applications in molecular sensing.3,4 The IDA relies on the competition between a dye indicator and an analyte for the same binding site of a receptor.2 When an analyte is introduced to a solution containing a host−indicator complex, the indicator is displaced by the analyte from the binding site. Usually, the free and bound indicators have different optical properties. On the basis of the displacement of the indicator by the analyte, a signal change is observed. Graphene (Gra) has been increasingly exploited as an outstanding quencher for fluorescence based on fluorescence resonance energy transfer (FRET) between a fluorescent dye and Gra in fluorescent sensing fields.5,6 Currently, fluorescent IDA (FIDA) has been widely used for potential applications in constructing a “switch−off−on” Gra fluorescent sensing platform.7 In a typical FIDA, a dye indicator is bound to a receptor first. A competitive analyte is then added into the sensing ensemble leading to the displacement of the indicator.6,7 Compared with the traditional method that label a dye aptamer on the surface of Gra, such an FIDA recognition approach, does not need the dye indicator to be covalently © XXXX American Chemical Society

Received: August 24, 2015 Accepted: November 16, 2015

A

DOI: 10.1021/acsami.5b07833 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces Scheme 1. Fluorescent Indicator Displacement Assay for Tadalafil (Tad) Using CX6−Gra against RhB

Figure 1. AFM images of Gra (A) and CX6−Gra (B). characterized by a JEM 2100 transmission electron microscopy (TEM, Tokyo, Japan) and a Nanoscope III atomic force microscopy (AFM). A Thermo Fisher Scientific Nicolet IS10 (Waltham, USA) Fourier transform infrared (FTIR) impact 410 spectrophotometer was used for the FTIR study. A Q50 thermogravimetric analysis (TGA, New Castle, USA) instrument was used for the TGA test from 25 to 800 °C in argon at a heating rate of 5 °C min−1. A Malvern Zetasizer Nano series was used for the ζ-potential measurements. Molecular Docking Study. The molecular docking procedure was according to our previously reported method.23 Crystal structures of CX6 (Scheme S2, ID: FEQYOQ) and tadalafil (ID: QUMAI) were obtained from Cambridge Crystallographic Data Centre (CCDC). The initial crystal structures were optimized by the Gaussian 03 program using molecular dynamics simulation. The optimized structures were used as a starting structure in the molecular docking study. The program of AutoDock4.2 with Lamarckian Genetic Algorithm (LGA) was used in this work. An initial population of 150 individuals was used for the docking studies. A maximum number of energy evaluations of 25 000 000 and a maximal number of generations of 27 000 were used as an end criterion. The probability of mutation and crossing-over was 0.02 and 0.8, respectively. The conformational search space implementing of a 60 × 60 × 60 grid was used for the docking study. A 0.375 Å spacing between each point was defined to cover the external surface and the internal cavity of the CX6 host. A total of 50 docking runs were performed. The solutions were

have been used to monitor tadalafil in drug analysis. Herein, a novel fluorescent IDA-based method for tadalafil sensing based on a competitive host−guest interaction between p-sulfonated calix[6]arene (CX6) and signal probe (Rhodamine B, RhB)/ target molecules (tadalafil) was developed by using CX6 functionalized Gra (CX6−Gra) as a receptor for the first time. As illustrated in Scheme 1, when RhB enters into the CX6 host its fluorescence is quenched by Gra. However, upon the presence of tadalafil to the performed CX6−Gra−RhB complex, the RhB molecules are displaced by tadalafil, leading to the recovery of the fluorescent signal of the dye, along with a “turn−on” fluorescent signal.

■

EXPERIMENTAL SECTION

Chemicals and Materials. The graphite oxide was obtained from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). pSulfocalix[6]arene (CX6, Scheme S1) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Rhodamine B (RhB), butyl rhodamine B (BRB) and safranine T (ST) were purchased from Shanghai Adamas Reagent Co., Ltd. (Shanghai, China). Deionized water (DW, 18 MΩ cm) was used for preparing aqueous solutions. Apparatus. A Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan) was used for fluorescence titration experiments at room temperature. The morphology of the obtained samples were B


Research Article

ACS Applied Materials & Interfaces

Figure 2. Fluorescence titrations of RhB (A, 10 μM, λex = 495 nm), ST (C, 10 μM, λex = 520 nm), and BRB (E, 10 μM, λex = 480 nm) upon successive addition of CX6 (up to 10 μM) in 0.1 M PBS (pH 7.0); fluorescence titration for the competitive displacement of RhB (B, 10 μM), ST (D, 10 μM), and BRB (F, 10 μM) from CX6 (10 μM) by tadalafil (up to 10 μM) in 0.1 M PBS (pH 7.0). The combined solution was mixed by vortexing well for 5 min and then tested. Additionally, the Gra was prepared with the similar procedure in the absence of CX6. Fluorescence Titration Experiments. A stock solution of dye (500 μM) and a stock solution of CX6 (500 μM) or CX6−Gra (1.0 mg mL−1) in DW were prepared. A stock solution of 500 μM tadalafil was prepared in acetonitrile as tadalafil has poor solubility in water. A final concentration of 10 μM dye solution was obtained by diluting the stock solution using 0.1 M pH 7.0 PBS. The CX6 or CX6−Gra was then gradually added until the dye fluorescence was quenched. For the competitive displacement of dye from the CX6 or CX6−Gra by tadalafil, the required amount of tadalafil was gradually added into the performed CX6−dye or CX6−Gra−dye complex. The combined solution was mixed by vortexing well for 5 min before the fluorescence was recorded. Control experiments were performed using Gra solution with the same procedure. Tadalafil Detection in Serum. Tadalafil detection in serum was performed using human serum. A stock solution of 500 μM tadalafil was prepared in acetonitrile, which was diluted to various concentrations by 0.1 M pH 7.0 PBS. The serum sample was diluted 50 times using 0.1 M pH 7.0 PBS. Then known amounts of tadalafil were added into the sample. Finally, this solution was used to detect tadalafil according to the procedure described above.

separated into clusters on the basis of their lowest RMSD and the best energy score value at the end of each run. From all the docking results, the lowest energy conformation was adopted as the binding mode of tadalafil/CX6 complex. Semiempirical method PM3 was used to calculate the binding free energy of the tadalafil/CX6 complex. Preparation of the CX6−Gra. The graphene oxide (GO) sheets was exfoliated from graphite oxide by ultrasonication in DW at room temperature for 1 h.23 The obtained brown GO aqueous suspension was stored at room temperature for further use. A wet-chemical approach was adopted to prepare the CX6−Gra composite rather than the traditional procedure using highly toxic hydrazine as reducing agent. In a typical experiment, 20 mL of 1.0 mg mL−1 CX6 aqueous solution was mixed with 20 mL of 0.5 mg mL−1 GO aqueous suspension. Then the mixture suspension was allowed for stirring at room temperature for 12 h. After that, a small amount of NaOH aqueous solution (1.0 M) was used to adjust the pH of the mixture to 11.0. Finally, the suspension was transferred to a flask and stirred at 90 °C in an oil bath for 5 h. After the reaction, the stable black dispersion of the CX6−Gra mixture was centrifuged at a relative centrifugal force (RCF) of 30600g. After washing with DW for three times, the resulting CX6−Gra composite was collected by lyophilization. C


Research Article


Figure 3. Fluorescence spectra of 10 μM RhB (A), BRB (C), and ST (E) upon successive addition of CX6 (up to 10 μM) and plots of 1/(F0 − F) versus 1/[CX6] for RhB (B), BRB (D), and ST (F).

■

RESULTS AND DISCUSSION Characterization of CX6−Gra. A wet-chemical route was adopted to prepare the CX6−Gra with excellent dispersibility in water, which was carried out by heating the GO suspension under strong alkali conditions in the presence of CX6. The obtained CX6−Gra could be stably dispersed in water even after removing free CX6 via high-speed centrifugation at a RCF of 30600g. After being stored for more than 6 months, there are no precipitates are observed in the CX6−Gra aqueous solution (Figure S1). As shown in Figure 1, the AFM characterizations of the Gra and CX6−Gra materials indicated that the thickness of CX6−Gra (1.3 nm) was higher than that of Gra (0.9 nm), demonstrating that grafting of CX6 on the surface of Gra results in the enhanced thickness. Recently, Chen et al. reported that phosphonated calix[8]arene forms ordered monolayer arrays on Gra, with their orientation controlled by the orientation of the hexagonal motif of the Gra.24 However, the CX6 here did not form ordered monolayer arrays on Gra. This may be due to the fact that the phosphonated calix[8]arene−Gra obtained by Chen et al. was directly exfoliated from graphite along with the self-assembly of

phosphonated calix[8]arene molecules onto the Gra while the present CX6−Gra was obtained by a wet-chemical route (heating the GO suspension under strong alkali conditions in the presence of CX6). On the other hand, it may be caused by the stronger negative charge of phosphate groups than that of −SO3−. The TEM characterizations of Gra and CX6−Gra were also obtained (Figure S2). However, the CX6 molecules on the TEM images cannot be observed. Moreover, the synthesized Gra and CX6−Gra materials were characterized by FTIR as shown in Figure S3, significant characteristics can be observed by comparing the FTIR spectra of CX6, CX6−Gra, and Gra. First, the typical peaks for −SO3− at 1160 and 1040 cm−1 both appeared in the FTIR spectra of native CX6 and the CX6−Gra composite, implying that CX6 molecules were grafted to the surface of Gra.14 Second, the peak value (3391 cm−1) of −OH in CX6 shifted to 3360 cm−1 in CX6−Gra, which was caused by the hydrogen bonding interactions between the remaining oxygen-containing groups of Gra and −OH groups of CX6.6 In addition, it has been reported that the composites of calixarenes and carbon materials (e.g., carbon nanotube, Gra) could be formed by π−π interactions and hydrogen bonding interD


Research Article


Figure 4. Fluorescence spectra of 50 μM tadalafil upon successive addition of CX6 (up to 10 μM) (A) and plots of 1/(F0 − F) versus 1/[CX6] (B).

actions.13−16 As shown in Figure S4, the prepared Gra and CX6−Gra materials were characterized by TGA analysis. For the pure Gra, the loss in mass (26%) was caused by the pyrolysis of the remaining oxygen-containing functional groups that have not been completely removed.25 An abrupt mass loss was presented in the CX6−Gra at a temperature of 450 °C, which was ascribed to the decomposition of CX6 molecules. The mass loss was approximately 46 wt % at a temperature of 600 °C. It can be estimated that approximately 20 wt % CX6 was grafted to the surface of Gra. Therefore, all these results demonstrated that the CX6 had successfully grafted to the surface of Gra. The ζ-potential measurements of the Gra and CX6−Gra materials were also obtained as shown in Figure S5. The average ζ-potential of Gra and CX6−Gra was −40.5 and −44.8 mV, respectively. The ζ-potential of CX6−Gra decreased approximately 4.3 mV compared with that of Gra, which was ascribed to the negative charge of −SO3−. Generally, all the ζpotentials were lower than −30 mV, suggesting that the colloidal stability of the Gra and CX6−Gra dispersions was very high. Fluorescence Titration. The fluorescence spectra of three dyes (RhB, BRB, and ST) in the presence of various concentrations of CX6 were investigated. Figure 2A shows the fluorescence titrations of RhB (10 μM, λex = 495 nm) upon successive addition of CX6 (up to 10 μM) in 0.1 M PBS at pH 7.0. The addition of CX6 caused the quenching of the fluorescence intensities of RhB solutions. Interestingly, the addition of tadalafil to the mixture of CX6 and RhB led to a successive reversion of the fluorescence intensity changes originally caused by the addition of the CX6 (Figure 2B). This may be attributed to the displacement of RhB by tadalafil from the CX6 host. The fluorescence titrations of BRB (10 μM, λex = 480 nm) and ST (10 μM, λex = 520 nm) were also obtained as shown in Figure 2C−F, which also indicated the displacements of BRB or ST by tadalafil from the CX6 host. Mechanism of the Competitive Host−Guest Interaction. The double reciprocal plots of 1/(F0 − F) versus 1/ [CX6] for RhB, BRB, and ST to CX6 were obtained (Figure 3), suggesting that these inclusion complexes were 1:1 stoichiometry.9 The binding constants (K) of the 1:1 RhB/CX6, BRB/ CX6, and ST/CX6 complexes were calculated to be 4.3 × 104, 2.6 × 104, and 4.1 × 104 M−1, respectively. The double reciprocal plot of 1/(F0 − F) versus 1/[CX6] for tadalafil to CX6 was also obtained (Figure 4). The binding constant of the 1:1 tadalafil/CX6 complex was calculated to be 2.2 × 105 M−1. The K value of tadalafil/CX6 complex was more than 5 times greater than those of RhB/CX6, BRB/CX6, and ST/CX6, which demonstrated the stronger binding of tadalafil with CX6

than those with RhB, BRB, and ST. Besides, the CX6/tadalafil inclusion complex was studied by molecular docking study. Generally, if the binding energy is more negative, the interaction between the host and guest will be stronger. As provided in Table S1, for a 1:1 inclusion complex of tadalafil and CX6, the lowest binding free energy (ΔG) was −7.18 kcal/ mol. From the following equation: ΔG = −RT ln K (R is the gas constant, T is the temperature), the K value of the tadalafil/ CX6 complex could be estimated to be 1.85 × 105 M−1, which is close to the value (2.2 × 105 M−1) obtained by the fluorescence method. As shown in Figure 5A, the docked

Figure 5. (A) Lowest energy tadalafil/CX6 docked complex for 1:1 host−guest stoichiometry (left is the side view, right is the top view); electrostatic forces (B, left is the top view of CX6, right is the top view of tadalafil/CX6 complex; red represents the strongest positively charged, blue represents the strongest negatively charged) and hydrophobic forces (C, left is the top view of CX6, right is the top view of tadalafil/CX6 complex; brown represents the strongest hydrophobic, blue represents the strongest hydrophilic) of tadalafil/ CX6 docked complex for 1:1 host−guest stoichiometry. E


Research Article


Figure 6. (A) Effect of increasing concentrations of CX6−Gra (concentrations ranging from 0 to 50 μg mL−1) on the fluorescence intensity of RhB (λex = 495 nm). RhB concentration was 10 μM. (B) Fluorescence spectra of the CX6−Gra−RhB complex via different concentrations of tadalafil (0−50 μM). RhB and CX6−Gra concentrations were 10 μM and 50 μg mL−1, respectively. The combined solution was mixed by vortexing well for 5 min and then tested. (C) Calibration curves of fluorescence intensity (CX6−Gra−RhB) vs tadalafil concentration. (D) Photographs of 10 μM RhB (a), 10 μM RhB+50 μg mL−1 CX6−Gra (b), and 10 μM RhB+50 μg mL−1 CX6−Gra+50 μM tadalafil (c) upon excitation under 365 nm UV light.

in fluorescence quenching was Gra. As shown in Figure 7, it is obvious that the fluorescence intensity of RhB was quenched by

conformation for the lowest energy of the 1:1 inclusion complex of tadalafil and CX6 implies that the partial inclusion of tadalafil molecule in the hydrophobic cavity of CX6. The indole and benzodioxole parts of the tadalafil molecule inserted into the cavity of the CX6 host. The results obtained from the docking studies reveals that π−π interactions, electrostatic interactions, and hydrophobic interactions are the main driving forces of the host−guest complex. First, the benzene rings on indole and the benzodioxole parts of the tadalafil molecule formed π−π interactions with the benzene rings of CX6. Second, significant electrostatic interactions formed between the positive part of the tadalafil molecule and the negative −SO3− of CX6 as shown in Figure 5B. Third, as presented in Figure 5C, there are strong hydrophobic interactions between tadalafil and the CX6 molecule. It is worth noting that the molecular simulation here was simplified. Actually, the influence of the water environment and the chemisorption of the CX6 to the Gra on the binding of the tadalafil molecule with the CX6 should be considered. These issues have not been clarified in the present study and the predicting binding interplay between the calixarene and the molecules should only be considered as a guide. Nevertheless, given that there is strong binding of the molecule, the lowest energy and common double cone confirmation of the CX6 is likely to be directed away from the surface of the Gra. Fluorescence Spectra Analysis. The fluorescence quenching performance of the CX6−Gra was investigated by using RhB as a dye indicator. As shown in Figure 6A, with the increasing of the concentration of CX6−Gra, the fluorescence intensity of RhB gradually decreased. A control experiments were carried out to verify that the predominant factor resulting

Figure 7. Fluorescence spectra of 10 μM RhB, 10 μM RhB in the presence of 10 μM CX6, and 10 μM RhB in the presence of 50 μg mL−1 CX6−Gra.

approximately 95% in the presence of the CX6−Gra composite. Whereas the addition of free CX6 led to a fluorescence quenching of approximately 7.5%, revealing that the energy transfer between the dye and Gra predominantly contributed to the fluorescence quenching of the dye. Other fluorescent dye indicators (BRB, ST) have also been studied and showed similar quenching effects (Figures S6 and S7). Here, we have concentrated more on RhB as it offers the strongest fluorescence signal. In contrast, as illustrated in Figure 6B, the successive addition of tadalafil to the performed CX6− Gra−RhB complex led to a successive reversion of the F


Research Article


Figure 8. (A) Effect of increasing concentrations of Gra (concentrations ranging from 0 to 70 μg mL−1) on the fluorescence intensity of RhB (λex = 495 nm). RhB concentration was 10 μM. (B) Fluorescence spectra of the Gra−RhB complex via different concentrations of tadalafil (0−20 μM). RhB and Gra concentrations were 10 μM and 70 μg mL−1, respectively. The combined solution was mixed by vortexing well for 5 min and then tested.

(20 μM) were added to the CX6−Gra−RhB complex aqueous solution. As shown in Figure S12A, the sildenafil and vardenafil showed negligible changes in fluorescence intensity. The interferences from common molecules present in human blood were also studied. As presented in Figure S12B, carbohydrates, protein, anionic surfactant, and some salts showed negligible interference. The concentration of the interferences including glucose, sucrose, ascorbic acid (AA), bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), Tween 20, NaCl, and KCl was 1.0 mM, whereas the concentration of tadalafil was only 20 μM. The changes in the fluorescence ratio (F − F0)/F0 of the CX6−Gra−RhB complex upon addition of the interferences are displayed in Figure 9. Upon addition of the interferences, the fluorescence

fluorescence of RhB. As shown in Figure 6D, photographs of 10 μM RhB (a), 10 μM RhB+50 μg mL−1 CX6−Gra (b), and 10 μM RhB+50 μg mL−1 CX6−Gra+50 μM tadalafil (c) upon excitation under 365 nm UV light were obtained, suggesting the displacement of RhB by tadalafil molecules. Control experiments have been performed to confirm that the observed fluorescence recovery is caused by the displacement of RhB by tadalafil from the CX6 host. As depicted in Figure 8, although the fluorescence quenching effect is significant for Gra, the quenched fluorescence is not “turned on” upon the addition of tadalafil. Thus, it can be concluded that the dye indicator formed inclusion complex with CX6−Gra first. Then it released form CX6−Gra upon the addition of tadalafil accompanied by a phenomenon of fluorescence “switch−off−on”. The observed fluorescence response can be used for quantitative detection of tadalafil. The calibration curves for tadalafil quantification were obtained and depicted in Figure 6C. The fluorescence ratio F/ F0 were proportional to the tadalafil concentrations. The linear response ranges of 1.00 to 23.00 μM and 23.00 to 50.00 μM with a detection limit of 0.32 μM (S/N = 3) was obtained. The corresponding regression equations were calculated as F/F0 = 0.15C (μM) + 1.02 and F/F0 = 0.033C (μM) + 3.76, respectively. When tadalafil was added to the performed CX6− Gra−BRB and CX6−Gra−ST complexes (Figures S8 and S9), the same fluorescence reversion was also observed. Photographs of BRB and ST in the absence and presence of CX6− Gra and BRB/ST-bound CX6−Gra with tadalafil upon excitation under 365 nm UV light were also obtained (Figures S10 and S11). Compared with that of cyclodextrins, the conformational flexibility of calixarenes is much larger.12 On the other hand, the sulfonate groups of p-sulfonated functionalized calixarenes make up an upper rim with negative charge. The negative charge was responsible for their preferential binding with cationic guests.12 Thus, calix[6]arene−Gra materials are expected to have wider applications than traditional cyclodextrin−Gra. Other analytical techniques including, HPLC with fluorescence,19 GC−MS,20 immunoassay,21 and electrochemical method22 have been used to detect the concentration of tadalafil. Compared with these approaches for tadalafil detection, the present method is very simple, convenient, and may be expanded to other applications. Selectivity and Analytical Application. Before the application, the selectivity of the proposed method for determination of tadalafil was investigated. The typical analytes (tadalafil, or sildenafil and vardenafil) of the same concentration

Figure 9. Relative fluorescence intensity is calculated by (F − F0)/F0, where F0 and F are the fluorescence intensity without and with the presence of tadalafil (20 μM), sildenafil (20 μM), vardenafil (20 μM), glucose (1 mM), sucrose (1 mM), Tween 20 (1 mM), BSA (1 mM), AA (1 mM), SDS (1 mM), NaCl (1 mM), and KCl (1 mM), respectively.

changes of the CX6−Gra−RhB complex enhanced selectively for tadalafil. However, nonsignificant fluorescence changes caused by the addition of other interferences. The high selectivity toward tadalafil beyond sildenafil and vardenafil may be due to the stronger π−π interaction between tadalafil and CX6 than that of sildenafil and vardenafil. The negligible interference from these molecules presented in human blood suggested that the molecular recognition between tadalafil and CX6 was stronger than those of the common interferences. Standard addition method was used to determine tadalafil in G



■

human serum (Table 1). The satisfactory apparent recovery revealed that the proposed fluorescent method could be expanded for determination of tadalafil in human blood.

added (μM)

founded (μM)

RSD (%)

recovery (%)

1 2 3

2.0 5.0 10.0

1.9 ± 0.094 5.2 ± 0.207 9.8 ± 0.488

4.95 3.98 4.98

95.0 104.0 98.0

■

CONCLUSIONS In conclusion, a simple and convenient fluorescent method based on a competitive host−guest interaction between CX6 and signal probe/target molecules using CX6−Gra as a receptor for the determination of tadalafil was developed. Because of the outstanding quenching performance of the Gra and the excellent molecular recognition ability of CX6, the constructed fluorescent sensing platform displays excellent analytical performance for the detection of tadalafil. The present proposed fluorescent approach provides a sensitive and selective instrument for monitoring the drug metabolite in pharmaceutical therapy. In addition, the inclusion complex of tadalafil and CX6 was studied by molecular docking to rationalize the experimental results. The molecular docking results implied that the complex of 1:1 host−guest stoichiometry had the lowest ΔG value of −7.18 kcal/mol and the driving forces including electrostatic interactions, π−π interactions, and hydrophobic interactions should be responsible for the formation of such inclusion complex.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b07833. Chemical structure and crystal structure of CX6, photograph of CX6−Gra dispersion, AFM, TEM, FTIR, TGA, and ζ-potential characterizations of Gra and CX6−Gra, quenching effects of CX6−Gra on BRB and ST, fluorescence recovery CX6−Gra−BRB and CX6−Gra−ST by tadalafil, photographs of BRB and ST in the absence and presence of CX6−Gra and with tadalafil, selectivity for the detection of tadalafil, and interaction energy between tadalafil and CX6 (PDF).

■

REFERENCES

(1) Yalow, R. S.; Berson, S. A. Immunoassay of Endogenous Plasma Insulin in Man. J. Clin. Invest. 1960, 39, 1157−1175. (2) Ghale, G.; Nau, W. M. Dynamically Analyte-Responsive Macrocyclic Host−Fluorophore Systems. Acc. Chem. Res. 2014, 47, 2150−2159. (3) Biedermann, F.; Uzunova, V. D.; Scherman, O. A.; Nau, W. M.; De Simone, A. Release of High-Energy Water as an Essential Driving Force for the High-Affinity Binding of Cucurbit[n]urils. J. Am. Chem. Soc. 2012, 134, 15318−15323. (4) Biedermann, F.; Vendruscolo, M.; Scherman, O. A.; De Simone, A.; Nau, W. M. Cucurbit[8]uril and Blue-Box: High-Energy Water Release Overwhelms Electrostatic Interactions. J. Am. Chem. Soc. 2013, 135, 14879−14888. (5) Mondal, A.; Jana, N. R. Fluorescent Detection of Cholesterol using β-Cyclodextrin Functionalized Graphene. Chem. Commun. 2012, 48, 7316−7318. (6) Mao, X.; Tian, D.; Li, H. p-Sulfonated Calix[6]arene Modified Graphene as a ‘Turn On’ Fluorescent Probe for L-Carnitine in Living Cells. Chem. Commun. 2012, 48, 4851−4853. (7) Nguyen, B. T.; Anslyn, E. V. Indicator−Displacement Assays. Coord. Chem. Rev. 2006, 250, 3118−3127. (8) Biedermann, F.; Nau, W. M. Noncovalent Chirality Sensing Ensembles for the Detection and Reaction Monitoring of Amino Acids, Peptides, Proteins, and Aromatic Drugs. Angew. Chem., Int. Ed. 2014, 53, 5694−5699. (9) Li, C. F.; Feng, J. X.; Ju, H. X. Supramolecular Interaction of Labetalol with Cucurbit[7]uril for its Sensitive Fluorescence Detection. Analyst 2015, 140, 230−235. (10) Praetorius, A.; Bailey, D. M.; Schwarzlose, T.; Nau, W. M. Design of a Fluorescent Dye for Indicator Displacement from Cucurbiturils: a Macrocycle-Responsive Fluorescent Switch Operating through a pKa Shift. Org. Lett. 2008, 10, 4089−4092. (11) Mutihac, L.; Lee, J. H.; Kim, J. S.; Vicens, J. Recognition of Amino Acids by Functionalized Calixarenes. Chem. Soc. Rev. 2011, 40, 2777−2796. (12) Dsouza, R. N.; Pischel, U.; Nau, W. M. Fluorescent Dyes and Their Supramolecular Host/Guest Complexes with Macrocycles in Aqueous Solution. Chem. Rev. 2011, 111, 7941−7980. (13) Chen, X. J.; Boulos, R. A.; Eggers, P. K.; Raston, C. L. pPhosphonic Acid Calix[8]arene Assisted Exfoliation and Stabilization of 2D Materials in Water. Chem. Commun. 2012, 48, 11407−11409. (14) Zhou, J.; Chen, M.; Diao, G. W. Calix[4,6,8]arenesulfonates Functionalized Reduced Graphene Oxide with High Supramolecular Recognition Capability: Fabrication and Application for Enhanced Host−Guest Electrochemical Recognition. ACS Appl. Mater. Interfaces 2013, 5, 828−836. (15) Eroglu, E.; Zang, W. Z.; Eggers, P. K.; Chen, X. J.; Boulos, R. A.; Haniff Wahid, M.; Smith, S. M.; Raston, C. L. Nitrate Uptake by pPhosphonic Acid Calix[8]arene Stabilized Graphene. Chem. Commun. 2013, 49, 8172−8174. (16) Chen, X. J.; Gibson, C. T.; Britton, J.; Eggers, P. K.; Haniff Wahid, M.; Raston, C. L. p-Phosphonic Acid Calix[8]arene Assisted Dispersion and Stabilisation of Pea-Pod C60@Multi-Walled Carbon Nanotubes in Water. Chem. Commun. 2015, 51, 2399−2402. (17) Giannitsas, K.; Perimenis, P. Phosphodiesterase-5 Inhibitors: Future Perspectives. Curr. Pharm. Des. 2009, 15, 3540−3551. (18) Badr-Eldin, S. M.; Elkheshen, S. A.; Ghorab, M. M. Inclusion Complexes of Tadalafil with Natural and Chemically Modified βCyclodextrins. I: Preparation and In-Vitro Evaluation. Eur. J. Pharm. Biopharm. 2008, 70, 819−827. (19) Farthing, C. A.; Farthing, D. E.; Koka, S.; Larus, T.; Fakhry, I.; Xi, L.; Kukreja, R. C.; Sica, D.; Gehr, T. W. A Simple and Sensitive HPLC Fluorescence Method for Determination of Tadalafil in Mouse Plasma. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2010, 878, 2891−2895. (20) Nikolaou, P.; Papoutsis, I.; Athanaselis, S.; Alevisopoulos, G.; Khraiwesh, A.; Pistos, C.; Spiliopoulou, C. Development and

Table 1. Detection of Tadalafil in Human Serum Samples (n = 3) sample

Research Article

AUTHOR INFORMATION

Corresponding Author

*C.-P. Li. Fax/Tel: +86-871-65031119. E-mail: lcppp1974@ sina.com. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21565029, 31160334) and the Natural Science Foundation of Yunnan Province (2012FB112, 2014RA022), People’s Republic of China. H


Research Article

ACS Applied Materials & Interfaces Validation of a GC/MS Method for the Determination of Tadalafil in Whole Blood. J. Pharm. Biomed. Anal. 2011, 56, 577−581. (21) Guo, J. B.; Xu, Y.; Huang, Z. B.; He, Q. H.; Liu, S. W. Development of an Immunoassay for Rapid Screening of Vardenafil and Its Potential Analogues in Herbal Products Based on a Group Specific Monoclonal Antibody. Anal. Chim. Acta 2010, 658, 197−203. (22) Yang, L.; Zhao, H.; Li, C.-P.; Fan, S. M.; Li, B. C. Dual βCyclodextrin Functionalized Au@SiC Nanohybrids for the Electrochemical Determination of Tadalafil in the Presence of Acetonitrile. Biosens. Bioelectron. 2015, 64, 126−130. (23) Yang, L.; Zhao, H.; Fan, S. M.; Zhao, G. F.; Ran, X.; Li, C.-P. Electrochemical Detection of Cholesterol Based on Competitive Host−Guest Recognition Using a β-Cyclodextrin/Poly(N-acetylaniline)/Graphene Modified Electrode. RSC Adv. 2015, 5, 64146−64155. (24) Chen, X. J.; Vimalanathan, K.; Zang, W. Z.; Slattery, A. D.; Boulos, R. A.; Gibson, C. T.; Raston, C. L. Self-assembled calixarene aligned patterning of noble metal nanoparticles on graphene. Nanoscale 2014, 6, 4517−4520. (25) Ran, X.; Yang, L.; Zhao, G. F.; Ye, H. Z.; Zhang, Y. Q.; Fan, S. M.; Xie, X. G.; Zhao, H.; Li, C.-P. Simultaneous Determination of Two Flavonoids Based on Disulfide Linked β-Cyclodextrin Dimer and Pd Cluster Functionalized Graphene-Modified Electrode. RSC Adv. 2015, 5, 60775−60785.

I


Fluorescent Detection of Tadalafil Based on Competitive HostâGuest

Recommend Documents

Fluorescent Detection of Tadalafil Based on Competitive HostâGuest