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A competitive strategy based on cucurbit[7] uril supramolecular interaction for simple and sensitive detection of dibucaine Yan Li, Chang-Feng Li, Li-Ming Du, Jian-Xia Feng, Hai-Long Liu, Yun-Long Fu

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Received date: 8 June 2014 Revised date: 29 August 2014 Accepted date: 3 September 2014 Cite this article as: Yan Li, Chang-Feng Li, Li-Ming Du, Jian-Xia Feng, Hai-Long Liu, Yun-Long Fu, A competitive strategy based on cucurbit[7]uril supramolecular interaction for simple and sensitive detection of dibucaine, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.09.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A competitive strategy based on cucurbit[7]uril supramolecular interaction for simple and sensitive detection of dibucaine Yan Li a, b, Chang-Feng Li b,*, Li-Ming Du a,*, Jian-Xia Feng a, Hai-Long Liu a and Yun-Long Fu b a

Analytical and Testing Center, Shanxi Normal University, Shanxi, Linfen 041004, PR China

b

School of Chemistry and Materials Science, Shanxi Normal University, Linfen 041004, PR China

Abstract In this work, the competitive interaction between dibucaine and three fluorescent probes (i.e., berberine, palmatine, and coptisine) for occupancy of the cucurbit[7]uril (CB[7]) cavity was studied by fluorescence spectra, UV-visible absorption spectra,

1

H NMR spectra, and

theoretical calculations in acidic aqueous solution. Based on the fluorescence enhancement of berberine, palmatine, and coptisine upon binding with CB[7], respectively, a series of fluorescence detection methods for dibucaine were proposed. At the optimized conditions, the fluorescence intensity of berberine-CB[7], palmatine-CB[7], and coptisine-CB[7] complexes showed negaitive correlation to the concentration of dibucaine, which led to a series of simple and sensitive fluorescence methods for the determination of dibucaine for the first time. The linear ranges obtained in the detection of the dibucaine were 0.018-3.34 µmol L-1, 0.032-4.47 µmol L-1, and 0.079-4.42 µmol L-1 with detection limits of 6.0 nmol L-1, 12.0 nmol L-1, and 25.0 nmol L-1, respectively. Moreover, the proposed method was successfully applied for the

*Corresponding author. Tel./Fax: +86-357-2057969. E-mail address: [email protected] ( C-F Li); [email protected] ( L-M Du ). -1-

determination of the drug in biological fluids. The competitive mode based on CB[7] superstructure provided a promising assay strategy for fluorescence detection in various potential applications. Keywords: Dibucaine; Spectrofluorometry; Competitive interaction; Supramolecular chemistry; Cucurbit[7]uril

1. Introduction

Dibucaine, 2-butoxy-N-(2-diethylaminoethyl)-4-quinolinecarboxamide hydrochloride (DC) (Scheme 1) has attracted great clinical interest for its 15 times more potent than procaine and five times than cocaine in producing local anaesthesia, but with considerably more poisonous property [1]. A number of assays have been reported for detection of DC in biological and pharmaceutical samples, including differential-pulse polarographic [1], chromatography [2-9], atomic absorption spectrometry [10] and spectrophotometry [11]. However, chromatography requires the use of organic solvents, expensive instruments, and time-consuming operating steps [2-9]. Though easier to operate, the reported method of spectrophotometry also need the use of haematoxylin in the presence of boric acid to give a reddish-violet chromogen fluorescent product, and showed low sensitivity and limited extending potential [11]. Some other methods are not sensitive enough [1, 10]. Therefore, simple and sensitive detection method is in urgent need for determination of DC especially in biological fluids. To the best of our knowledge, no fluorescence analyst method is available for the detection of DC. Supramolecular chemistry provides a possibility for selective detection of DC due to the steric effect of macrocyclic compounds. Supramolecular complex formation of macrocyclic

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compounds is widely utilized as molecular-scale devices, sensors, fluorescent probes due to the amphiprotic properties of macrocyclic compounds [12–14]. Specially, the cucurbit[n]urils (CB[n], n = 5-8, 10), which are comprised of n glycoluril units bridged by 2n methylene groups, possess considerable negative charge density in their carbonyl-fringed portals for the binding of metal ions and cationic organic compounds. Meanwhile the extremely nonpolarizable cavity preferably accommodates hydrophobic moieties [15–18]. The CB[7] host (Scheme 1) has been of particular interest in fluorescent assay since its remarkable ability to form host-guest complexes with organic molecules [19–26]. Herein, we employ berberine hydrochloride (BER), palmatine hydrochloride (PAL), and coptisine (COP) as signal probes for the fluorescence detection of DC in biological fluids samples (Scheme 2). When DC was added into the aqueous solution of CB[7], there was no distinct fluorescence change. However, dramatic increase in fluorescence intensity was observed when BER, PAL, or COP was added to CB[7] solution, respectively. As such, when DC was mixed with CB[7]-berberine, CB[7]-palmatine, or CB[7]-coptisine complexes, the significant decrease of fluorescence intensity can be observed, respectively. The fluorescence intensity showed a negative correlation to the concentration of DC with a linear calibration plot in the ranges of 0.018-3.34, 0.032-4.47, and 0.079-4.42 µmol L-1, with detection limits of 6.0 nmol L-1, 12.0 nmol L-1, and 25.0 nmol L-1 for the above three complexes, respectively. Moreover, the designed method was successfully applied in the detection of DC in urine samples, which showed promising application in various practical sceneries. 2. Experimental

2.1. Materials and reagents Berberine hydrochloride (BER), palmatine hydrochloride (PAL), coptisine (COP), and DC -3-

were obtained from the Chinese National Institute for the Control of Pharmaceutical. CB[7] was prepared and characterized according to the reported procedure [17, 18]. CB[7] stock solutions of 1.0 mmol L-1 were prepared by dissolving CB[7] in a 100 mL volumetric flask with doubly-distilled water. All the stock standard solutions were stable for several weeks at room temperature. All other reagents used were of analytical grade, and double-distilled water was used thoroughly.

2.2. Apparatus The fluorescence measurements were performed on a Cary Eclipse spectrofluorometer (America). The slit widths of both excitation and emission monochromators were set to 5 nm. All measurements were performed in a standard 10 mm path-length quartz cell set to a temperature of 25.0 ± 0.5 ◦C. Absorption spectra were measured on a U-3010 UV-visible spectrophotometer (Hitachi, Japan). 1H NMR spectra were obtained using a Bruker AV-600 MHz spectrometer (Switzerland) in D2O buffered with DCl (0.10 mol L-1). Molecular modeling calculations were optimized at the B3LYP/6-31 G(d) level of density functional theory with the Gaussian 03 program.

2.3. Spectra measurement procedure

The fluorescence measurements were carried out with the excitation wavelength of 345, 344, and 358 nm in the absence or presence of DC solution, respectively. In a 10 mL colorimetric flask, 0.5 mL 0.1 mmol L-1 CB[7] solution, 0.5 mL 0.1 mmol L-1 probe molecular solution (i.e. BER, PAL, or COP), followed by 1.0 mL of 1.0 mol L-1 hydrochloric acid, and a given concentration of DC standard solution or sample solution were sequentially added, respectively. The mixture was diluted to the volume with water and shaken for 15 min at room temperature.

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2.4. Analysis of spiked human urine

Urine samples were handled according to the previous protocol [27]. Briefly, each urine sample (10.0 mL) was placed into a centrifuge tube, spiked with 1.0 mL drug stock solution and then centrifuged at 5000 rpm for 5 min. The clear supernatant of the spiked urine sample was extracted by solid phase extraction (SPE) using 001× 7 strong-acid cation exchange resin. The procedure was as follows: 1 mL of the spiked urine sample was transferred into the SPE cartridge (with the resin height being 1 cm and the diameter 1 cm). After allowing the sample to pass through to waste under gravity, the resin was washed with 1 mL 1% sodium hydroxide solution and 2 mL double-distilled water (amino acids can be removed). The filtrate was discarded. Finally, the analyte was eluted into a colorimetric flask with 4 mL of methanol/water (1:1, v/v) and then the general procedure was followed. 3. Results and discussion 3.1. Spectral characteristics Because the same trends showed for three fluorescent probes (i.e., BER, PAL or COP), the following research used the BER as example. The UV-visible spectra of DC and BER showed absorption bands at 202, 246, 321 nm and 228, 263, 345 nm, respectively (Fig. 1A, curve a and b). After the addition of CB[7] into DC or BER solution, a slight hypochromicity was observed (Fig. 1A, curve c and d). This should be attributed to the inclusion of DC or BER by CB[7] and produce supramolecular complexes. However, a increase in absorption spectra was observed upon addition of DC into BER-CB[7] complex solution (Fig. 1A, curve e). This should be attributed to the BER molecule expelled from the CB[7] cavity because of the introduction of DC. The solution of BER alone showed undetectable fluorescent emission, meanwhile, DC also -5-

exhibited very weak native fluorescence in acidic aqueous solution (Fig. 1B, curve a and b). After the addition of CB[7] into DC solution, no distinct change of fluorescence was observed (Fig. 1B, curve c). However, a dramatic increase in fluorescence intensity was observed upon addition of CB[7] in BER solution (Fig. 1B, curve d). This should be attributed to the inclusion of BER by CB[7] to change the space structure or conformation of BER and produce a fluorescent complex. Interestingly, the addition of DC to the mixture of CB[7] and BER led to significant decrease of fluorescence intensity (Fig. 1B, curve e). Furthermore, with the increasing concentration of DC, the fluorescence intensity became smaller (Supporting information, Fig.S1, Fig.S2, Fig.S3). The fluorescence intensity decrease to the amount of added DC led to a series of simple and sensitive fluorescence methods for the determination of DC.

3.2. Interaction mechanism of the fluorescent probe

Although the fluorescent emission of pure BER, PAL, and COP was very weak or undetectable, the fluorescence intensity greatly enhanced after they entered the hydrophobic cavity of CB[7], which showed a good linear relationship between 1/F and 1/cCB[7], indicating the existence of a 1:1 complex. The binding constant (K) for these complexes were determined to be 9.57 × 104, 4.26 × 104, and 1.86 ×105 L mol -1 , respectively [19–21]. When DC was added to CB[7]-complexes solution, DC could compete to occupy the CB[7] cavity with BER, PAL and COP, respectively. Due to the greater KDC–CB[7] (1.1 ± 0.2 × 107 L mol -1) value [28] than KCB[7]–BER, KCB[7]–PAL, and KCB[7]–COP, which showed the stronger binding of DC with CB[7], BER, PAL, and COP in CB[7] cavity was displaced by DC. As a result, the fluorescent intensity of CB[7]-BER, CB[7]-PAL, CB[7]-COP complex weakened. The formation of CB[7]-DC inclusion complex could be confirmed by 1H NMR spectroscopy -6-

(Fig. 2). Compared with the proton resonance of the unbound DC molecule (Fig. 2A), the resonance of protons H1`, H2`, H3`, and H4` of the bound DC in the 1H NMR spectrum of CB[7]-DC complex experienced a progressively up-field shift (Fig. 2B), indicating that CB[7] binds selectively to the protonated oxygen-terminal butyl residues due to cooperative hydrophobic and ion-dipol interactions. Because of the excellent matched size and morphology, butyloxy residues was enclosed in the cavity of CB[7] more tightly than triethylammonium groups in acidic aqueous solution. The resonance of protons H3 of DC experienced a slightly down-field shift, indicating this part of the molecule was located just outside the carbonyl portal of the CB[7] host. Molecular modeling calculations were optimized at the B3LYP/6-31G(d) [29] level of density functional theory [30, 31] using the Gaussian 03 program. The results confirmed the partial inclusion of DC in the hydrophobic cavity of CB[7] (Fig. 3). It can be seen from molecular simulation that both butyloxy group and quinolyl group were protonated in acidic solution, giving the cationic form of DC. This indicated that, in the energy-minimized structure, butyloxy group of the molecule located inside the host, however, quinolyl group of the molecule located just outside the carbonyl portal of the CB[7] host.

3.3. Analytical performance From the Supporting information (Fig.S1, Fig.S2, Fig.S3), in the solution of 5.0 µmol L-1 BER-CB[7], PAL-CB[7], and COP-CB[7] complexes, the plots of fluorescence intensity vs the concentration of DC showed a series of linear relationship in the ranges of 0.018-3.34 µmol L-1, 0.032-4.47 µmol L-1, and 0.079-4.42 µmol L-1, respectively. The linear regression equations were I = -90.42c + 602.08, I = -80.23c + 626.08, and I = -86.69c + 819.81 (c denotes the

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concentration of DC in µmol L-1) with the correlation coefficients of 0.9997, 0.9991, and 0.9995, and the detection limits of 6.0 nmol L-1, 12.0 nmol L-1, and 25.0 nmol L-1 at 3σ for the same order, respectively. A comparison of the results obtained by the proposed method with those obtained by the other spectroscopic approaches shows the sensitive is satisfactory (Table 1). Prior to the application of the proposed fluorescence method in analysis of the drug in human urine samples, the effect of common ingredients in human urine on the determination of DC was examined. The criterion for interference was fixed at a ±5% variation in the average fluorescence intensity calculated for the established level of DC. The results showed no interference of the common ingredients in urine samples. However, the components such as cysteine, alanine, phenylalanine, and valine could change the fluorescence intensity of the complex to a certain degree. Hence, they should be separated prior to the determination. 3.4. Analytical application

Due to the high sensitivity, good selectivity and stability shown in the proposed fluorescent method, the method was applied in the determination of the DC in spiked samples of human urine. The results are presented in Table 2 (using the CB[7]-BER complex as signal probe). The standard deviations obtained from the proposed method were less than 1.40%. The recoveries examined with a standard addition method were in the range of 98.0-103.5%. 4. Conclusion

In this work, the supramolecular systems of CB[7] were designed for the first time in the determination of DC. The CB[7]-BER, CB[7]-PAL, and CB[7]-COP complexes were -8-

indentified to have a greatly enhanced fluorescent emission due to strong coplanar and rigidity of BER, PAL, and COP, respectively. Since the interaction of DC with CB[7] was stronger than BER, PAL, and COP, the CB[7]-BER, CB[7]-PAL, and CB[7]-COP superstructures would be disintegrated upon addition of DC considering the thermodynamic factor, which led to a significant fluorescence reduce, respectively. Based on the competitive mode, a series of novel fluorescence methods for the determination of DC were established with high sensitivity, cost-effective, and easy operation, which was successfully applied in biological fluids. The interaction mechanism between the DC and the CB[7] was also confirmed that the butyloxy group of the DC molecule located inside the host in acidic aqueous solution via UV-visible absorption spectra, the 1H NMR spectrum and the theoretical calculations. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21171110) and the foundation of Shanxi Normal University (No. ZR1210). Helpful suggestions by anonymous referees are also gratefully acknowledged.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.xxxx.xx.xxx.

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Scheme 1 Structure of DC and CB[7].

Scheme 2 Competitive recognition of CB[7] to DC against BER, PAL, or COP. Fig. 1. (A) UV-visible spectra of (a) DC, (b) BER, (c) DC+CB[7], (d) BER+CB[7] and (e) BER+CB[7]+DC. The concentrations of BER, CB[7] and DC are 10.0 µmol L-1, respectively, pH=1. (B) Fluorescence spectra of (a) BER, (b) DC, (c) DC+CB[7], (d) BER+CB[7] and (e) BER+CB[7]+DC. The concentrations of BER, CB[7] and DC are 5.0, 5.0, and 3.34 µmol L-1, respectively, pH=1.

Fig. 2. 1H NMR (600 MHz) spectra of (A) DC and (B) DC in the presents of CB[7] in D2O buffered with DCl (0.10 mol L-1).

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Fig. 3. Energy-minimized structure of CB[7]-DC complex in the ground state using balls and tubes for rendering the atoms. Color codes for DC and CB7: oxygen, red; nitrogen, blue; carbon, dark gray; hydrogen, gray.

Table 1 Comparison with other proposed methods for the determination of DC. Table 2 Fluorescence determination of DC in spiked urine (n = 5, p = 95%).

Scheme 1 Structure of DC and CB[7]

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Scheme 2 Competitive recognition of CB[7] to DC against BER, PAL, or COP

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Fig. 1. (A) UV-visible spectra of (a) DC, (b) BER, (c) DC+CB[7], (d) BER+CB[7] and (e) BER+CB[7]+DC. The concentrations of BER, CB[7] and DC are 10.0 µmol L-1, respectively, pH=1. (B) Fluorescence spectra of (a) BER, (b) DC, (c) DC+CB[7], (d) BER+CB[7] and (e) BER+CB[7]+DC. The concentrations of BER, CB[7] and DC are 5.0, 5.0, and 3.34 µmol L-1, respectively, pH =1.

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Fig. 2. 1H NMR (600 MHz) spectra of (A) DC and (B) DC in the presents of CB[7] in D2O buffered with DCl (0.10 mol L-1).

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Fig. 3. Energy-minimized structure of DC-CB[7] complex in the ground state using balls and tubes for rendering the atoms. Color codes for DC and CB7: oxygen, red; nitrogen, blue; carbon, dark gray; hydrogen, gray.

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Table 1 Comparison with other proposed methods for the determination of DC.

Technique

Linear range -1

(µmol L )

LOD

Application

Reference

-1

(µmol L )

Differential-pulse polarographic

1-100

0.5

Pharmaceutical ointment

[1]

HPLC

0.004-0.079

----

----

[3]

HPLC

0.132-13.2

0.034

Human urine

[4]

GC

13.2-105.3

4.42

Biological fluidsand tissues

[5]

GC-MS

1.32-263.2

0.26

Plasma; Urine

[6]

LC-MS

----

0.013

Human serum

[7]

CS-LC-MS

0.0263-2.63

0.0086

Plasma

[8]

HIEKC

105.3-289.5

2.63

Commercial ointment

[9]

Atomic absorption spectrometry

152.6-952.6

10.97

Pharmaceutical formulation

[10]

99.99-999.9

2.13

Ointment

[10]

Spectrophotometry

13.2-78.96

4.42

Pharmaceutical preparations

[11]

Spectrofluorimetry CB[7]/BER

0.018-3.34

0.006

Human urine

This work

CB[7]/PAL

0.032-4.47

0.012

Human urine

This work

CB[7]/COP

0.079-4.42

0.025

Human urine

This work

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Table 2 Fluorescence determination of DC in spiked urine (n = 5, p = 95%) Samples

Spiked urine DC added

DC found

Recovery(%)

(µg mL )

(µg mL )

±S.D.a

0.1

0.098

98.0 ± 1.27

Urine 2

0.2

0.197

98.5±1.25

Urine 3

0.4

0.414

103.5 ±1.30

Urine 4

0.8

0.807

100.9±0.80

Urine 5

1.0

1.025

102.5±1.40

–1

a

–1

Average of five determination

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Highlights ►The fluorescence method was used for determination of dibucaine for the first time. ► The competitive strategy was designed for determination of dibucaine. ►The occupancy of the cucurbit[7]uril cavity was studied by spectroscopy and

calculations.

►The proposed method showed good sensitivity for detection of dibucaine in biological fluids.

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*Graphical Abstract (for review)

Graphical Abstract:

A competitive strategy based on cucurbit[7]uril supramolecular interaction for simple and sensitive detection of dibucaine By Yan Li , Chang-Feng Li , Li-Ming Du , Jian-Xia Feng , Hai-Long Liu and Yun-Long Fu

The competitive interaction between dibucaine (DC) and three fluorescent probes (i.e., berberine (BER), palmatine (PAL), and coptisine (COP)) for occupancy of the cucurbit[7]uril (CB[7]) cavity was observed and used for simple and sensitive detection of dibucaine.