A novel water-soluble quantum dot-neutral red fluorescence resonance energy transfer probe for the selective detection of megestrol acetate

Article abstract of DOI:10.1039/c4nj01443j

A novel water-soluble beta-cyclodextrin (β-CD)-functionalized ZnS quantum dot (QD)-neutral red (NR) fluorescence resonance energy transfer (FRET) probe for the selective determination of the concentration of megestrol acetate in river water has been developed. The water-soluble and low-toxicity β-CD-functionalized ZnS QDs were first synthesized, and their characterization was confirmed by transmission electron microscopy and infrared, UV-vis and fluorescence emission spectra. The NR molecule can enter the cavity of the β-CD anchored onto the surfaces of the ZnS QDs in its neutral form, forming the FRET probe. Compared with other steroid hormones, the probe can selectively recognize megestrol acetate at a lower concentration level. The possible underlying mechanism of the probe with nine steroid hormones was discussed in detail. The fluorescence quenching fractions of the probe presented a satisfactory linearity with the concentrations of megestrol acetate, and its limit of detection was calculated to be 0.0083 μM. Coupled with sample pretreatment procedures, the probe has been applied to the determination of megestrol acetate in river water. The average recoveries of megestrol acetate in the spiking levels of 0.001 μM-10 μM ranged from 97% to 110% with a relative standard deviation below 15%, which was similar to those for HPLC or MS techniques.

Full text of DOI:10.1039/c4nj01443j

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A novel water-soluble quantum dot–neutral red
fluorescence resonance energy transfer probe for
the selective detection of megestrol acetate†
Cite this:DOI: 10.1039/c4nj01443j
Li-Yun Wang, Ling-Yu Dong, Luan Chen, Ya-Bing Fan, Jing Wu, Xiang-Feng Wang
and Meng-Xia Xie*
A novel water-soluble beta-cyclodextrin (b-CD)-functionalized ZnS quantum dot (QD)–neutral red (NR)
fluorescence resonance energy transfer (FRET) probe for the selective determination of the concentration
of megestrol acetate in river water has been developed. The water-soluble and low-toxicity b-CD-
functionalized ZnS QDs were first synthesized, and their characterization was confirmed by transmission
electron microscopy and infrared, UV-vis and fluorescence emission spectra. The NR molecule can enter
the cavity of the b-CD anchored onto the surfaces of the ZnS QDs in its neutral form, forming the FRET
probe. Compared with other steroid hormones, the probe can selectively recognize megestrol acetate at
a lower concentration level. The possible underlying mechanism of the probe with nine steroid hormones
was discussed in detail. The fluorescence quenching fractions of the probe presented a satisfactory
linearity with the concentrations of megestrol acetate, and its limit of detection was calculated to be
0.0083 mM. Coupled with sample pretreatment procedures, the probe has been applied to the
determination of megestrol acetate in river water. The average recoveries of megestrol acetate in the
spiking levels of 0.001 mM–10 mM ranged from 97% to 110% with a relative standard deviation below 15%,
which was similar to those for HPLC or MS techniques.
Received (in Montpellier, France)
28th August 2014,
Accepted 28th October 2014
DOI: 10.1039/c4nj01443j
www.rsc.org/njc
probes for genetic target strand detection,21 and the high speci-
ficity of hybridization between QD–DNA probes and the target
1. Introduction
Quantum dots (QDs) are popular nanostructured materials that strand with a complementary sequence forms the basis for the
possess unique optical and electronic properties, such as size- detection of DNA.22
dependent emission, high quantum yield and simultaneous
Cyclodextrins (CDs) are cyclic oligosaccharides in a, b or g
resistance to photobleaching.1–3 Thus, QDs have generated a forms, which can act as molecular hosts for forming inclusion
great deal of attention over the past two decades and are complexes with various guest molecules due to their special
recognized as a new type of prospective fluorescent probes that molecular structure consisting of a hydrophobic internal cavity
are useful for molecular recognition,4 metal ion detection,5,6 and a hydrophilic external surface. These CD-functionalized
biological macromolecule interaction,7,8 cell imaging,9–11 and QD particles were successfully utilized to selectively and rever-
other applications. However, the practical applications of the sibly control the analyte-induced fluorescence change in the
QDs for the specific determination of target analytes are QDs.23–27 Li and Han reported the synthesis of the water-
dependent on the modification of the QD surfaces.
soluble CdSe/ZnS QDs by a simple sonochemical method using
Early research mainly focused on small organic molecules, a-, b- and g-CDs as surface-modifiers, and the obtained probes
primarily thiols, as the modification agents of the QD have high sensitivity for the determination of p-nitrophenol
surfaces,12–15 and in recent years, numerous functional groups and 1-naphthol.23 They also reported that a-CD- or b-CD-
or biological macromolecules have been successfully utilized.16–20 functionalized CdSe/ZnS QDs are useful for the highly enantio-
For example, DNA or RNA fragments can act as the recognition selective fluorescent recognition of amino acids.28 Furthermore,
reporters and are connected to the QD surface to form fluorescent b-CD-functionalized CdSe/ZnS QDs have been used for the optical
sensing of 1-adamantanecarboxylic acid and 4-hydroxytoluene
and the chiroselective sensing of D,L-phenylalanine and
Analytical & Testing Center of Beijing Normal University, Beijing 100875,
D,L-tyrosine.29 An optical biosensor for the determination of
P. R. China. E-mail: xiemx@bnu.edu.cn; Fax: +86-10-58800076; Tel: +86-10-58807981
amantadine based on FRET between b-CD-functionalized
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4nj01443j
CdTe QDs and rhodamine B has been constructed.30 It has
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also been found that the addition of ferrocene derivatives to the
b-CD-anchored QD system would result in a significant decrease
in the band-edge emission via a photoinduced electron transfer
mechanism, while upon the addition of adamantine to the
system, a high luminescence response can be observed.31
Although the development of fluorescent probes based on the
system has been impressive, the scientific community is still
actively attempting to develop additional selective and sensitive
approaches for practical applications.
FRET between donor and acceptor chromophores has been
extensively utilized to explore the interaction mechanism
between biomacromolecules and their ligands32 and to develop
novel approaches for the determination of target analytes.33–36
Amine-modified QD605 and Cy5-labeled oligodeoxynucleotides
were chosen as the FRET pair and were encapsulated in
lipoplexes; upon excitation at 405 nm, the QD-FRET mediated
Cy5 emission suggested compact and intact lipoplexes, and the
disappearance of QD-FRET-mediated Cy5 emission indicated
the dissociation of the lipoplexes.37 Efficient FRET from molecular
beacon-modified CdTe QDs to graphene oxide has also been
reported, and the strong interaction between the single-stranded
DNA of the molecular beacon loop structure and graphene oxide
was combined to establish a novel selective and sensitive platform
for the fluorescence-quenching detection of DNA.38 Due to the
sensitive and selective properties of the FRET-based systems
for analyte determination purposes, the continued development
of more pragmatic systems is the main branch of analytical
chemistry.
Steroid hormones play an important role in maintaining
life, immune regulation, skin diseases and birth control. These
hormones may be released into the environment by illegal sewage
effluent and can potentially interfere with the normal function of
the endocrine system, which would affect both reproduction and
development in wildlife.39–41 Therefore, monitoring the residues
of the steroid hormones in the environment is important. The
conventional techniques for the analysis of steroid hormones are
Scheme 1 (A) Schematic illustration for the preparation of the b-CD
modified ZnS QDs. (B) Formation of the FRET system between b-CD-
modified ZnS QDs and neutral red NR (i); the possible fluorescence
quenching mechanism of the CD-QD–NR FRET fluorescence probe
which was induced by 9H (ii); competitive assay of the other eight steroid
hormones (1–8H) and NR on the cavities of the fluorescence probe (iii).
hormones (see Scheme 1B(ii) and (iii)). The conditions of the
FRET system have been optimized, and the behaviours of the
fluorescence quenching properties for the FRET system
induced by various types of steroid hormones have been
systematically investigated. An interaction mechanism of the
FRET system with different steroid hormones has been
proposed. Consequently, a simple, rapid and low-cost analytical
method based on a FRET fluorescence probe for the determina-
tion of the concentration of megestrol acetate in river water
samples has been developed.
2. Experimental methods
2.1. Chemicals and apparatus
HPLC, GC–MS and LC–MS–MS.40,42–44 However, some disadvan- All chemicals obtained from commercial suppliers were used
tages of these approaches exist, such as the tedious nature of without further purification. Zinc acetate dehydrate (Zn(Ac)2ꢀ
these procedures, the difficultly in performing the experiments 2H2O, 99.0%) and sodium sulphide nonahydrate (Na2Sꢀ9H2O,
and the high cost of equipment. Optical analysis is considered a 98.0%) were obtained from Sinopharm Chemical Reagent Co.,
more convenient and alternative technique due to its inherent Ltd (Beijing, China). Neutral red (NR) and testosterone (98.0%)
simplicity and high sensitivity. Recently, there have been several were purchased from Acros Organics (New Jersey, USA).
reports of rapid detection methods for steroid hormones. For Progesterone (98.0%) was obtained from Aladdin Chemistry
example, commercially available rapid ELISA kits were validated Co., Ltd (Shanghai, China). 4-Androstene-3,17-dione(98.0%),
for the quantification of oestrogens in sewage effluent samples,45 stanozolol (98.0%) and 17a-hydroxyprogesterone (97.0%) were
and a unique FRET-based aptasensor was constructed for the obtained from Dr Ehrenstorfer GmbH (Augsburg, Germany).
determination of 17 beta-estradiol using a quantum dot bio- Medroxyprogesterone acetate (96.0%) and megestrol acetate
conjugate as a nano-biosensor and a fluorescence-labelled anti-17 (96.0%) were obtained from TCI Chemicals (Shanghai, China).
beta-estradiol aptamer as a bio-recognition molecule.46
Estrone (96.0%) and 17-beta-estradiol (96.0%) were obtained
In this report, a b-CD-ZnS QD–NR FRET probe has been from J&k Chemical. Ltd (Beijing, China). C18 (40–60 mm) was
developed (see Scheme 1). Mono-6-thio-b-CD was prepared by a obtained from Merck (Darmstadt, Germany). Acetonitrile and
simple two-step synthesis and then conjugated with the ZnS QD hexane were of HPLC-reagent grade and were provided by
particles (see Scheme 1A). The dye NR could bind to the cavity Sinopharm Chemical Reagent Beijing Co., Ltd (Beijing, China),
of b-CD, and the FRET process could occur between b-CD- and other chemicals and reagents were of analytical grade and
functionalized ZnS QDs and NR (see Scheme 1B(i)). The devel- were obtained from Beijing Chemical Factory (Beijing, China).
oped b-CD-ZnS QD–NR FRET system was sensitive to steroid Ultrapure water was prepared using a Milli-Q water system
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(18.3 MO cmꢁ1) from a Millipore purification system (Barnstead ultrapure water to prepare a stock solution with a concentration
Corp., Boston, USA).
of 0.01 mol Lꢁ1. NR was dissolved in ultrapure water to prepare a
The SPE cartridge (6 mL) and sieve plates were purchased stock solution with a concentration of 3.5 ꢂ 10ꢁ3 mol Lꢁ1. Nine
from Jiruisen (Beijing, China). Separation was carried out using types of steroid hormones were dissolved in ethanol to prepare
solid-phase extraction equipment with 12-port vacuum manifolds stock solutions with a concentration of 4.0 ꢂ 10ꢁ3 mol Lꢁ1
.
(Supelco, Bellefonte, PA, USA). Concentration was performed
2.4. Fluorescence emission spectra
using an EVA 30A nitrogen-blowing instrument (Polytech. Co.
Ltd Beijing, China).
To study the pH effect on the FRET system, the mixed solution of
b-CD-functionalized ZnS QDs and NR was diluted with buffer
solutions at different pH values (pH 2.5, 4.5, 6.5, 8.5 and 10.5). The
final concentrations of b-CD-functionalized ZnS QDs and NR were
2.2. Preparation and characterization of the b-CD-
functionalized ZnS QDs
The mono-6-thio-b-CD was prepared according to the procedures 300 mM and 1.8 mM, and the fluorescence spectra were collected.
described previously.26,47 Briefly, to a three-necked flask, Zn(Ac)2ꢀ
To study the effect of the NR concentrations on the FRET
2H2O (0.11 g, 0.5 mmol), mono-6-thio-b-CD (0.85 g, 0.75 mmol) system, a series of concentrations of NR (0, 0.2, 0.4, 0.6, 0.8, 1.0,
and water (50 mL) were added, and the mixture solution was 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, and 2.4 mM) were mixed with b-CD-
stirred until the components dissolved. The pH of the solution functionalized ZnS QDs (final concentration 300 mM) in a buffer
was adjusted to 11 using 1 M NaOH, and then the mixture was solution (pH 8.5), and then the fluorescence spectra were collected.
refluxed for 20 min after the removal of air by N2 bubbling for
To study the response of the b-CD-QD–NR FRET fluores-
15 min at room temperature. Thereafter, 5 mL of Na2Sꢀ9H2O cence probe to steroid hormones, the nine steroid hormones
(0.12 g, 0.5 mmol) was added to the solution, and the solution were mixed with the probe in a buffer solution (pH 8.5). The
was stirred for 20 min under N2 protection; the solution was final concentrations of the b-CD-functionalized ZnS QDs and
then aged at room temperature for 6 h to form the b-CD- NR were 300 mM and 1.8 mM, respectively, and for the steroid
functionalized ZnS QDs. For purification, the obtained product hormones, the concentrations were 10 mM, 60 mM and 180 mM.
was precipitated with ethanol three times and then separated Subsequently, their fluorescence spectra were collected.
by centrifugation and dried under vacuum. The prepared b-CD-
functionalized ZnS QDs were refrigerated at 4 1C and were quite NR FRET fluorescence probe, ten kinds of common ions (Ca2+
stable and water soluble. 3-Mercaptopropionic acid (MPA)- Cd2+, Mn2+, Co2+, Mg2+, Na+, K+, Fe3+, Fe2+ and Cu2+) were
modified ZnS QDs were prepared using similar procedures. mixed with the probe in buffer solution (pH 8.5), respectively.
To investigate the effects of coexisting ions on the b-CD-QD–
,
For characterization, the infrared spectra of b-CD and b-CD- The final concentrations of b-CD-functionalized ZnS QDs and
functionalized ZnS QDs were collected using a Nicolet Nexus NR were 300 mM and 1.8 mM, individually, and the concen-
670 FT-IR spectrometer (Thermofisher, Madison, WI, USA) in tration for each ion was 180 mM, and then, their fluorescence
the range of 4000–400 cmꢁ1 with 64 scans at a resolution of spectra were collected.
4 cmꢁ1. The morphology of the b-CD-functionalized ZnS QDs
The effect of concentration on the steroid hormone fluores-
was characterized using a Tecnai F20 transmission electron cence emission intensities of the b-CD-QD–NR FRET fluores-
microscope (FEI) operated at a voltage of 200 kV (Hillsboro, cence probe was determined as follows. The mixtures of
USA). UV-vis absorption spectra of b-CD-functionalized ZnS megestrol acetate and the probe in buffer solutions (pH 8.5)
QDs and NR in a phosphate-buffered solution (pH 8.5) were with final concentrations of 0, 0.25, 0.5, 1.0, 5.0, 10, 20, 40 and
acquired using a SPECORD 200 spectrophotometer (analytikjena, 60 mM megestrol acetate were prepared, and their fluorescence
Gena, Germany) in the range of 220–700 nm with a slit of 2 nm. spectra were collected. Similarly, the mixture solutions of
Fluorescence emission spectra of b-CD-functionalized ZnS QDs the probe and various concentrations of the other steroid
and NR in a phosphate-buffered solution (pH 8.5) were obtained hormones (0, 60, 120, 180, 240, 300 and 360 mM) were prepared,
using a HORIBA Jobin Yvon FluoroMax-4 fluorometer (JY, Paris, and their spectra were individually collected.
France) over ranges of 350–550 nm and 500–700 nm at an
excitation wavelength of 300 nm. The slit width for both excitation collected using a 370 nm filter and scanned in the range of
and emission was 5 nm. 380–700 nm at an excitation wavelength of 300 nm. The slit
The quantum yield of the b-CD-functionalized ZnS QD width for both excitation and emission was 5 nm.
particles was directly determined in ultrapure water using a Control experiments for the interaction of NR and steroid
HORIBA Jobin Yvon FluoroMax-4 fluorometer with a F-3029 hormones were conducted via the following methodology. The NR
The above-mentioned fluorescence emission spectra were
Integrating Sphere accessory (JY, Paris, France).
solution was diluted to 1.8 mM with pH 8.5 buffer solution, and the
mixture of NR and b-CD was diluted to concentrations of 1.8 mM
and 300 mM with buffer solution (pH 8.5) Then, a series of mixture
2.3. Preparation of stock solution
The phosphate buffer solution (10 mmol Lꢁ1) was adjusted to solutions (pH 8.5) of NR (1.8 mM) and b-CD (300 mM) and steroid
pH 2.5, 4.5, 6.5, 8.5 and 10.5 using NaOH or H3PO4. b-CD- hormones (megestrol acetate (60 mM) and other steroid hormones
functionalized ZnS QDs were dissolved in ultrapure water to (180 mM)) were prepared. Their fluorescence spectra were recorded
prepare stock solutions with a concentration of 0.01 mol Lꢁ1 in the range of 500–700 nm at the excitation wavelength of 300 nm,
(estimated according to ZnS molecules). b-CD was dissolved in and the slit width for excitation and emission was 5 nm.
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Control experiments for the interaction of b-CD-functionalized purified and enriched by solid-phase extraction (SPE) via the
ZnS QDs and steroid hormones followed the procedure outlined following procedure.41,48,49
below. The b-CD-functionalized ZnS QDs were diluted to 300 mM
The C18 (1.0 g) sorbents were used to fill a 6 mL SPE
with buffer solution (pH 8.5). The mixture solutions of b-CD- cartridge and then conditioned with 10 mL of n-hexane,
functionalized ZnS QDs (300 mM) and megestrol acetate (60 mM) 10 mL of acetonitrile and 10 mL of ultrapure water. Thereafter,
and other steroid hormones (180 mM) were prepared. Their the spiked water sample (320 mL) was loaded onto the SPE
corresponding fluorescence spectra were collected in the range cartridge at a flow rate of 4–5 mL minꢁ1, and the cartridge was
of 350–550 nm at an excitation wavelength of 300 nm.
dried under vacuum. The SPE cartridge was eluted with 5 mL of
acetonitrile, and the elution was evaporated using a gentle
stream of nitrogen gas. The residues were dissolved with
0.1 mL of ethanol and 1 mL of buffer solution (pH 8.5), and
the b-CD-QD–NR FRET fluorescence probe was added; then, the
solution was diluted to 3.2 mL with buffer solution. Each
sample was assayed five times (n = 5), and the corresponding
fluorescence spectra were collected.
2.5. Fluorescence lifetime measurements
The NR solution was diluted to 1.8 mM with buffer solution
(pH 8.5). The mixture solutions of b-CD-functionalized ZnS QDs
(300 mM) and NR (1.8 mM) were prepared with pH 8.5 buffer.
A series of mixed solutions for b-CD-functionalized ZnS QDs
(300 mM) and NR (1.8 mM) and the steroid hormones (60 mM for
megestrol acetate and 180 mM for other steroid hormones) were
prepared with buffer solution (pH 8.5). Fluorescence lifetime
measurements were carried out by the time-correlated single-
photon counting (TCSPC) method using a Horiba Jobin Yvon
Fluorocube (JY, Paris, France).
3. Results and discussion
3.1. Characterization of the b-CD-functionalized ZnS QDs
Scheme 1A gives details of the preparation and grafting of the
mono-6-thio-b-CD onto the surface of ZnS QD particles. The
obtained b-CD-functionalized ZnS QDs have been characterized
by FTIR spectra, transmission electron microscopy, UV-vis
absorption and fluorescence emission spectroscopic methods.
No typical absorption band was observed in the FTIR spectra
2.6. Spiking recoveries of megestrol acetate in river water
samples
River water samples were collected from local rivers in Beijing
and filtered prior to analysis. Five spiking levels of megestrol of ZnS QDs from 4000 to 500 cmꢁ1 50–52
and the IR spectra of
,
acetate (0.001, 0.01, 0.1, 1.0 and 10 mM) were added to 320 mL water-soluble ZnS QDs usually reflect the absorption bands
of river water samples. Then, the spiked water samples were of their modifier. Fig. 1A shows the transmission FTIR spectra
Fig. 1 (A) FTIR spectra of b-CD and b-CD-modified ZnS QDs. (B) The TEM image of b-CD modified ZnS QDs. (C) Spectral overlapping between b-CD
modified ZnS QDs and NR. a and a0 lines represent the fluorescence emission band and the UV-vis absorption band of b-CD-modified ZnS QDs,
respectively; b and b0 lines represent the fluorescence emission band and the UV-vis absorption band of NR, respectively. (D) The cationic form and the
neutral form of neutral red (NR) in aqueous solution.
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Fig. 2 (A) The fluorescence spectra of the b-CD-modified ZnS QDs (300 mM) and NR (1.8 mM) in pH 8.5 buffer solution (a, b), respectively; the
fluorescence spectra of the mixture of NR and b-CD-modified ZnS QDs (1.8 mM + 300 mM) in pH 8.5 buffer solution (c), excited at l = 300 nm. (B) The
fluorescence decay curves of NR (1.8 mM) in the absence (a) and the presence (b) of b-CD-modified ZnS QDs (300 mM), monitored at l = 564 nm.
(C) The fluorescence intensities of the b-CD-QD–NR FRET fluorescence probe at pH 2.5, 4.5, 6.5, 8.5 and 10.5, respectively, excited at l = 300 nm.
(D) The fluorescence emission spectra of the b-CD-QD–NR FRET system at various concentrations of NR (from a to j, the concentrations of NR were
0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 and 2.4 mM, respectively), excited at l = 300 nm.
of b-CD and b-CD-functionalized ZnS QDs. For the spectra of at 278 nm can be attributed to the benzene ring, and the band at
b-CD, the broad band at 3390 cmꢁ1 and the band at 1629 cmꢁ1 454 nm arises from the conjugated three-ring system of the NR
arise from the O–H stretching and asymmetric vibrations, molecule). The fluorescence emission band of NR was at 614 nm
respectively. The band at 2926 cmꢁ1 corresponds to the when excited at l = 300 nm. There was a remarkable overlapping
asymmetric stretching vibration (na) of the C–H bond, and the between the fluorescence emission band of the QDs (donors)
strong band at 1155 cmꢁ1 and the bands at 1084 and 1030 cmꢁ1 and the UV absorption band (454 nm) of the NR (acceptor),
can be ascribed to the asymmetric glycosidic vibration na(C–O–C) indicating that they would potentially provide an efficient
and the coupled stretching vibration n(C–C and C–O), respec- fluorescence energy transfer from the QDs to the dye NR if the
tively. It can been seen from the FTIR spectra of the b-CD- NR can enter the cavity of the b-CD on the surfaces of the QDs.
functionalized ZnS QDs that the spectral features and peak
The quantum yield of the b-CD-functionalized ZnS QDs was
positions of the main bands roughly resemble those of b-CD, determined to be 22.5%, which indicated that they can be used
which indicates that the b-CD has been successfully grafted onto for determination purposes.54
the surfaces of the ZnS QDs.
3.2. Establishment of the b-CD-QD–NR fluorescence FRET
probe
Fig. 1B shows the high-resolution transmission electron
microscopy image of the water-soluble b-CD-functionalized
ZnS QDs. It can be seen from the image that the diameters of
3.2.1. The b-CD-QD–NR FRET system. When the b-CD-
the particles range from 2 to 4 nm, and the sizes of the particles functionalized ZnS QDs were mixed with the dye NR in basic
are virtually identical, indicating that the b-CD-functionalized buffer solution, FRET could be observed between them. As
ZnS QDs are monodispersed and uniform in water.
shown in Fig. 2A, the fluorescence emission intensities
The UV-vis absorption and fluorescence emission spectra of (at 435 nm) of the ZnS QDs slightly decreased, and a strong
the b-CD functionalized ZnS QDs are shown in Fig. 1C. The fluorescence emission band suited at approximately 564 nm
maximum absorption and emission bands of the b-CD function- appeared, which may be the blue-shifted band of NR. It is
alized ZnS QDs were at 270 nm and 435 nm, respectively, observed that the fluorescence emission band of the free NR
demonstrating that grafting the b-CD onto the QDs does not was very weak (at 614 nm).
significantly influence the optical properties of the QDs.
For a control, 3-mercaptopropionic acid (MPA)-capped ZnS
Neutral red (NR) is a photoactive phenazine dye that generally QDs were prepared, and the optical properties of the mixture of
exists in two molecular forms in aqueous solution, both an MPA-capped ZnS QDs and NR were determined. The results
acidic and a neutral form53 (see Fig. 1D). As shown in Fig. 1C, showed that the FRET phenomenon cannot occur between
the UV-vis spectrum of NR has two absorption bands (the band these complexes (see Fig. S1, ESI†). The FRET between the
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Fig. 3 The chemical structures of the nine steroid hormones.
b-CD-functionalized ZnS QDs and NR illustrated that the NR
3.2.3. Optimization of the concentration of the NR in the
molecule entered the cavity of the b-CD on the surface of the ZnS probe. The fluorescence emission spectra of the b-CD-QD–NR
QDs, decreasing the distance between these molecules. The blue FRET system at different concentrations of NR were measured
shift of the fluorescence emission band for the NR probably in the buffer solution at pH 8.5 (see Fig. 2D). It can be seen that
originated from a conformational change after NR interacted the fluorescence intensities of the ZnS QDs (at 435 nm) slightly
with the b-CD and from alterations under the environmental decreased with increasing concentrations of NR, while those of
conditions after NR entered the hydrophobic cavity.53
NR (at 564 nm) significantly increased. When the concentration
Fig. 2B shows the decay curves of NR before and after NR of NR increased to 1.8 mM (the concentration of the b-CD-
entered the cavity of the b-CD on the surface of ZnS QDs functionalized ZnS QDs was 300 mM), the fluorescence intensity
(monitored at l = 564 nm), and the decay profiles were well of the NR reached its maximum and did not change further
fitted with a three-component exponential function, I(t) = a1 by an additional increase in the concentration of NR. The
exp(ꢁt/t1) + a2 exp(ꢁt/t2) + a3 exp(ꢁt/t3).55 The average fluores- b-CD-functionalized ZnS QD FRET system concentrations
cence lifetimes of NR were calculated to be 3.62 ꢂ 10ꢁ11 and were thus set at 300 mM for b-CD-functionalized ZnS QDs and
1.15 ꢂ 10ꢁ9 s. It can be seen that the lifetime of NR has 1.8 mM for NR.
substantially increased after NR enters the cavity, which further
3.3. Response of the b-CD-QD–NR FRET fluorescence probe to
supports the occurrence of FRET between b-CD-functionalized
steroid hormones
ZnS QDs and NR. As expected, the surface-anchored b-CD
on ZnS QDs still retained their host capability of including
molecular guests in their hydrophobic cavities.
3.3.1. Specificity of the probe to megestrol acetate. The
influence of nine steroid hormones, including 4-androstene-
3.2.2. The effects of pH on the properties of the probe. 3,17-dione (1H), testosterone (2H), progesterone (3H), stanozo-
Fig. 2C shows the effects of pH (2.5, 4.5, 6.5, 8.5 and 10.5) on lol (4H), 17a-hydroxy-progesterone (5H), medroxy-progesterone
the fluorescence emission intensity of NR in the b-CD-QD–NR acetate (6H), estrone (7H), 17-beta-estradiol (8H) and megestrol
FRET probe system. It can be seen that the fluorescence acetate (9H) was analysed. Their structures are shown in Fig. 3.
intensities of NR were relatively low under acidic conditions
The fluorescence intensity of the b-CD-QD–NR FRET probe
(pH 2.5 and 4.5), and the intensities increased as the pH of has been investigated at three concentration levels (10, 60 and
the buffer solution was increased to 6.5 and 8.5. However, the 180 mM), and the results are shown in Fig. 4. It can be seen that
intensity decreased when the pH reached 10.5. Conversely, the fluorescence intensities of the probe (at 564 nm) were
the pH of the phosphate buffer solution has nearly no effect significantly quenched upon increasing the concentration of
on the fluorescence intensities of b-CD-functionalized ZnS QDs. the megestrol acetate, while they were nearly unchanged when
The NR existed in its neutral form under basic conditions the concentrations of the other eight steroid hormones were
(as shown in Fig. 1D), and the results indicated that the neutral lower than 60 mM. This result provided an opportunity for us to
form of NR was favoured to enter the cavity of the b-CD and selectively determine the megestrol acetate at a lower concen-
form the FRET system. Thus, the optimal pH condition was tration level. However, the fluorescence intensities of the probe
selected as 8.5.
can be obviously quenched by the other eight steroid hormones
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the b-CD-QD–NR FRET probe was different from that for the
other eight hormones.
3.3.2. Interaction mechanism of the probe and steroid
hormones. To explore the fluorescence quenching mechanism
of the b-CD-QD–NR FRET probe, the average fluorescence lifetimes
of NR (t) for the probe after interacting with the steroid hormones
have been determined individually. The results indicated that the
lifetime of the NR in the probe for 9H was 1.08 ꢂ 10ꢁ9 s, which
was similar to that of the NR (1.15 ꢂ 10ꢁ9 s) prior to interacting
with 9H (see Fig. S4A, ESI†). This lifetime was much greater than
the fluorescence lifetime of NR (3.62 ꢂ 10ꢁ11 s) in the free state
(in buffer solution). This result illustrated that the 9H did not
displace the NR from the cavity of the b-CD on the surface of the
ZnS QDs after interacting with the probe and that the fluorescence
quenching of the probe may originate from the combination of 9H
and the NR in the cavity of the b-CD, forming complexes that have
no fluorescence.
Fig. 4 The fluorescence intensities (at 564 nm) of the b-CD-QD–NR
FRET fluorescence probe before and after interacting with various con-
centrations of nine steroid hormones, respectively, in the buffer solution
(pH 8.5). The concentrations of the steroid hormones were 10, 60 and
180 mM, respectively, b-CD-modified ZnS QDs (300 mM), NR (1.8 mM),
excited at l = 300 nm. The results were the average of three repeats.
After interacting with the other eight steroid hormones (1H
to 8H, 180 mM), the fluorescence lifetimes of NR (t ꢂ 10ꢁ11 s)
were 7.24, 8.99, 7.81, 8.14, 13.0, 6.84, 9.23 and 8.62 (Fig. S4B,
ESI† shows the representative decay curves of NR for the probe
at high concentration levels (see Fig. 4, and Fig. S2, the ESI† before and after interacting with the 5H), and these lifetimes
shows the representative fluorescence quenching spectra of the were significantly lower than that of the NR (1.15 ꢂ 10ꢁ9 s)
probe by 5H), which may potentially be utilized to monitor the prior to interaction with the steroid hormones and much closer
total content of the steroid hormones.
to that of the NR in the free state. It has been inferred that the
It was interesting to note that the megestrol acetate (9H) can NR molecule may be partly removed from the cavity of the b-CD
obviously quench the fluorescence intensities of the QDs at by these steroid hormones at higher concentration levels,
435 nm for the probe, and the other eight hormones (1H to 8H) which leads to the interruption of the FRET process and causes
have little effect on them at low concentration levels (10 and the reduction of the fluorescence emission intensities at 564 nm
60 mM). Conversely, these steroid hormones can cause the and the fluorescence enhancement of the QDs (at 435 nm)
enhancement of the fluorescence emission bands for the (see Fig. S2 and S3, ESI†).
QDs to various extents when the concentration of the other
For the confirmation of the above suppositions, control
hormones reaches 180 mM (see Fig. S3, ESI†). This pheno- experiments have been performed (see Fig. 5A). The fluorescence
menon and different fluorescence quenching behaviours of spectra of the NR solution and its mixture with a b-CD solution
the nine hormones with respect to the probe have implied that were collected, and the results showed that the fluorescence
the interaction mechanism between the megestrol acetate and emission intensity of the mixture of NR and b-CD was obviously
Fig. 5 (A) Control experiments for the interaction of NR and steroid hormones. The NR column represents the fluorescence intensity of individual NR
(1.8 mM); the NR + CD column represents the fluorescence intensity of NR (1.8 mM) after the addition of the b-CD (300 mM) solution; 1–9H columns
represent the fluorescence intensity of NR after mixing with the b-CD solution and the nine steroid hormones (megestrol acetate (60 mM) and other steroid
hormones (180 mM)), respectively. All fluorescence spectra were monitored at l = 564 nm and excited at l = 300 nm. (B) Control experiments for the
interaction of b-CD-modified ZnS QDs and steroid hormones. QDs column represents the fluorescence intensity (at 435 nm) of the b-CD-modified ZnS
QDs (300 mM), 1–9H columns represent the fluorescence intensity (at 435 nm) of b-CD-modified ZnS QDs after the addition of various steroid hormones
(1–9H) (megestrol acetate (60 mM) and other steroid hormones (180 mM)), respectively, excited at l = 300 nm. The results were the average of three repeats.
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higher than that of the free NR solution. This implied that the 3.4. Application of the b-CD-QD–NR FRET fluorescence probe
NR molecule entered the cavity of the b-CD. When the steroid for the determination of megestrol acetate in river water
hormones (1H–8H) were added to the mixture solution indivi- samples
dually, the fluorescence intensities of the mixture solution
decreased, but they were still higher than that of the free NR.
This result suggests that the NR molecule is partly displaced by
3.4.1. Relationship of the fluorescence intensities for the
probe with the concentrations of megestrol acetate. As discussed
above, the b-CD-QD–NR FRET fluorescence probe was sensitive
the steroid hormones. However, after interaction with the steroid
and specific to megestrol acetate at a lower concentration level,
hormone 9H, the fluorescence intensity of the mixture was
and therefore, the determination method for megestrol acetate
significantly decreased and was lower than those of the mixture
concentration described herein has been investigated and
and the free NR solution, which illustrated that 9H combined
developed.
with the NR molecule and formed a complex. The results of the
Fig. 6A shows the fluorescence quenching spectra of the
control experiments were similar to those of the interactions
probe with various concentrations of megestrol acetate in the
between the b-CD-QD–NR FRET probe and the steroid hormones,
range of 0–60 mM. It can be seen that the fluorescence inten-
and it has been verified that above suppositions are rational.
sities of the probe have been gradually decreased upon increasing
Similarly, the influence of the steroid hormones on the
the concentrations of the megestrol acetate, and their relation-
ship has been calculated based on the Stern–Volmer equation,57
as follows:
fluorescence intensities of the b-CD functionalized QDs has
also been investigated. The results showed that the megestrol
acetate (9H) can significantly quench the fluorescence of the
b-CD-functionalized QDs (at 435 nm), while the other eight
steroid hormones have nearly no effect on the fluorescence
intensities of the QDs (see Fig. 5B). This control experiment
illustrated that the fluorescence quenching of the b-CD-QD–NR
FRET probe (at 435 nm) may originate from the combination
of 9H with the QDs. Therefore, it can be concluded that the
megestrol acetate can form a complex with the NR molecule
and combine with the ZnS QDs in the probe, which interrupts
the FRET process and induces fluorescence quenching of
the probe.
The structure of megestrol acetate was different from that of
the other eight steroid hormones (see Fig. 3). For megestrol
acetate, the carbonyl group connected to ring A was conjugated
with the two double bonds of rings A and B, forming a larger
conjugated system. Therefore, the oxygen atom of the carbonyl
group would carry more negative charge and would enhance its
hydrogen bonding capability with the amino group of the NR
molecule or combine with the Zn2+ on the surface of the QDs.
For the other steroid hormones, the carbonyl group of ring A
was only conjugated with one double bond in ring A (1H, 2H,
3H, 5H and 6H), the hydroxyl group on ring A was conjugated
with the benzene ring (7H and 8H), or an imidazole ring
connected to ring A (4H). These functional groups of the steroid
hormones do not have enough electronegativity to combine
with the QDs or NR molecule, while they can enter the cavity of
the b-CD and replace the NR molecule at high concentration
levels.
I0
I
¼ 1 þ kQt0½Qꢃ ¼ 1 þ KSV½Qꢃ
(1)
where I0 and I represent the fluorescence intensities of the probe
in the absence and presence of megestrol acetate, [Q] is the
concentration of the megestrol acetate and t0 is the average
lifetime of the probe in the absence of megestrol acetate. The
quantities kQ and Ksv are the quenching rate constant and Stern–
Volmer quenching constants, respectively.
As shown in Fig. 6B, the curve for the fluorescence quenching
fractions of the probe versus the concentrations of megestrol
acetate presents satisfactory linearity in the concentration
ranging from 0.25 mM to 60 mM. The linear regression equation
was y = 0.0514x ꢁ 0.00124 with a regression coefficient (r) of
0.999, which indicated that the b-CD-QD–NR FRET fluorescence
probe can be utilized with confidence to determine the megestrol
acetate concentration. The limit of detection (LOD) for the probe
was evaluated using 3s/S and was found to be 0.0083 mM, where s
is the standard deviation of the blank signal, and S is the slope of
the linear calibration plot.58
Major ions and trace metals are usually present in aquatic
systems.56 In order to observe the influences of these ions on
the determination of megestrol acetate, the effects for 10 kinds
of common ions (Ca2+, Cd2+, Mn2+, Co2+, Mg2+, Na+, K+, Fe3+,
Fe2+ and Cu2+) on the b-CD-QD–NR FRET fluorescence probe
have been investigated (see Fig. S5, ESI†), individually. As
shown in Fig. S5 (ESI†), the fluorescence intensities of the
probe (at 564 nm) were nearly unchanged upon adding various
ions, which demonstrated that the common coexisting ions in
water did not interfere with the results for the determination of
the megestrol acetate.
Fig. 6 (A) The fluorescence quenching spectra of the b-CD-QD–NR
FRET fluorescence probe induced by various concentrations of megestrol
acetate (from a to i: 0, 0.25, 0.5, 1.0, 5.0, 10, 20, 40 and 60 mM) in buffer
solution (pH 8.5), b-CD-modified ZnS QDs (300 mM), NR (1.8 mM), excited
at l = 300 nm. (B) The Stern–Volmer plot for the fluorescence quenching
fractions (at l = 564 nm) of the probe versus the concentrations of
megestrol acetate. The results were the average of three repeats.
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The quenching rate constant kQ of the probe (NR) was the data shown in Table 1 that the limit of quantification (LOQ)
calculated to be 4.47 ꢂ 1013 L molꢁ1 sꢁ1 (t0 = 1.15 ꢂ 10ꢁ9 s) can reach 1.0 nmol, and the standard deviations would become
according to the above Stern–Volmer curve, which was approxi- very large upon decreasing the spiking levels, which cannot
mately three orders of magnitude higher than the limiting guarantee the accuracy of the determination results. In this
diffusion constant Kdif of the biomolecule (Kdif = 2.0 ꢂ case, the LOD and LOQ of the developed method were nearly
1010 L molꢁ1 ꢁ1).59,60 The results illustrated that a specific equal to those of previous reports for HPLC-MS49 or GC-MS48
s
interaction between the probe and megestrol acetate has methods, and were significantly lower than those of HPLC
occurred, and their fluorescence quenching mainly arose from approaches.63 As the data shown in Table 1, the satisified
static quenching by forming a complex59,61 between the probe spiking recoveries and RSD have been obtained in the concen-
and megestrol acetate.
tration range between 1.0 nM and 10 mM, which can be
The binding number (n) of megestrol acetate on the probe considered as the detection range of the megestrol acetate for
was calculated according to the following eqn (2), where KA is the developed method, and was much better than those of
the binding constant. The results showed that n = 0.85, which previous reports.49,63 The results illustrated that the method is
indicated that the ratio of probe and megestrol acetate in the sensitive and can be utilized for the rapid determination of
complex was approximately 1 : 1.
megestrol acetate in river water samples.
ꢀ
ꢁ
I0 ꢁ I
lg
¼ lg KA þ n log½Qꢃ
(2)
I
4. Conclusions
A novel beta-cyclodextrin functionalized ZnS quantum dot–
neutral red (NR) fluorescence resonance energy transfer probe
for the determination of megestrol acetate concentrations in
river water samples has been developed. The synthesis of the
water-soluble and low toxicity beta-cyclodextrin-functionalized
ZnS quantum dots was first reported in this paper. The dye
neutral red in its neutral form can enter the cavity of beta-
cyclodextrin anchored onto the surface of ZnS quantum dots,
forming an efficient fluorescence resonance energy transfer
fluorescence system. The interaction mechanisms of the
obtained probe with nine types of steroid hormones have been
investigated, and the results showed that megestrol acetate
induced the fluorescence quenching of the fluorescence reso-
nance energy transfer probe by forming complex, which was
different from the mechanisms for other steroid hormones.
The fluorescence resonance energy transfer fluorescence probe
was specific for megestrol acetate at low concentration levels,
and the developed method was specific, rapid and sensitive.
This methodology can determine the megestrol acetate con-
centrations in river water samples at the nanomolar level when
combined with the reported sample pretreatment procedures
and is equivalent to high performance liquid chromatography
or mass spectrometry techniques.
In this case, the binding constant (KA) of the complex can be
obtained from the slope of the curve based on the following
eqn (3), and the calculated apparent KA was 9.52 ꢂ 104 L molꢁ1
.
The large value of KA illustrated that the megestrol acetate
molecule has a high ability to bind with the probe, inducing its
significant fluorescence quenching, which further supported
the mechanism deduced in Section 3.3.2.
I0
ꢁ1
ꢁ1
¼ 1 þ KA ½Qꢃ
(3)
I0 ꢁ I
3.4.2. Spiking recoveries of megestrol acetate in river
water. The content of the steroid hormones in river water was
generally very low, so sample pretreatment procedures for
purification and enrichment were needed. Usually, the enrich-
ment factor for HPLC and MS methods was approximately
1000.41,48 Different spiking levels of megestrol acetate with
concentrations ranging from 0.001 mM to 10 mM in the river
water samples were performed, and the samples were then
enriched and purified using SPE procedures.41,48 Thereafter,
the concentrations of the residues were determined using
the developed b-CD-QD–NR FRET fluorescence probe, and the
results are summarized in Table 1. It can be seen from the data
in Table 1 that the average recoveries of megestrol acetate
ranged from 97 to 110%, with relative standard deviations
(RSD) below 15%, which satisfied the demands of residue
analysis.62 Considering the enrichment factor, theoretically,
the limit of detection (LOD) for the established method was
0.083 nM (the enrichment factor was 100). It can be noted from
Acknowledgements
This work was financially supported by the national science
and technology support program (2012BAK26B04).
Notes and references
Table 1 Analytical results for the detection of megestrol acetate levels in
river sample (n = 5)
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