A ratiometric fluorescent system for carboxylesterase detection with AIE dots as FRET donors

Article abstract of DOI:10.1039/c5cc04771d

A ratiometric fluorescent system for CaE detection with AIE dots as the FRET donors was designed. Upon enzymatic reaction, electrostatic interaction between the cationic TPE-N⁺ dots and the enzymatic reaction product - the negatively charged fluorescein molecules - allows the FRET process to proceed, thus affording the ratiometric fluorescence CaE assay.

Full text of DOI:10.1039/c5cc04771d

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A ratiometric fluorescent system for
carboxylesterase detection with AIE dots
Cite this:DOI: 10.1039/c5cc04771d
as FRET donors†
Received 10th June 2015,
Accepted 7th July 2015
Yinglong Wu, Shuailing Huang, Fang Zeng,* Jun Wang, Changmin Yu, Jing Huang,
Huiting Xie and Shuizhu Wu*
DOI: 10.1039/c5cc04771d
www.rsc.org/chemcomm
A ratiometric fluorescent system for CaE detection with AIE dots as efficiency by using the ACQ fluorophores as donors in nanoparticle-
the FRET donors was designed. Upon enzymatic reaction, electrostatic based FRET systems. Contrary to conventional fluorophores,
interaction between the cationic TPE-N+ dots and the enzymatic some organic compounds are only highly emissive when aggre-
reaction product – the negatively charged fluorescein molecules – gated due to the restricted intramolecular rotation/vibration,
allows the FRET process to proceed, thus affording the ratiometric which was referred to as ‘‘aggregation-induced emission’’ (AIE).5 To
fluorescence CaE assay.
date, a variety of AIE fluorophores have attracted widespread
interest in chemical sensors, biological imaging and optoelectronic
Fluorescent sensors are attractive and versatile tools for both devices. However, AIE dots are rarely adopted as donors in FRET
analytical sensing and optical imaging because of their high systems,5o–r which can avoid the limitations of ACQ-fluorophore-
sensitivity, fast response and technical simplicity.1 Many fluor- based donors and would be beneficial for nanoparticle-based
escent sensors employ increase or decrease in a single emission systems. Therefore, it is our primary interest to investigate the
intensity as the sensing signal that responds to the target employment of AIE dots as donors in the nanoparticle-based
analyte(s).2 However, a single fluorescence signal is readily FRET systems.
interfered by external factors and this sensing mode is sometimes
Carboxylesterase (CaE) is a group of enzymes that catalyze
problematic for precise analysis. While ratiometric sensing, which the hydrolysis of fatty acid esters into acids and alcohols with
involves the simultaneous measurement of two fluorescence water. Due to its broad substrate tolerance, CaE are widely used
signals at different wavelengths followed by the calculation of in organic synthesis and industrial production.6 It is also one
their intensity ratio, was devised to circumvent these unfavorable of the main enzymes involved in the detoxification of organo-
effects. Currently, several mechanisms have been exploited to phosphorus (OP) compounds and serves as a determinant of
realize ratiometric detection,3 and the fluorescence resonance individual sensitivity to these agents.7 Recently, it was discovered
energy transfer (FRET) process has been widely adopted for that human plasma carboxylesterase could be a novel serologic
ratiometric detection because of its facile control as a sensing biomarker candidate for hepatocellular carcinoma.8 And the
mode and its basis on well-established theory.3c–e
apparent CaE activity/level in human plasma (colorimetric
With the development of nanoparticles, more and more method, determined with 1-naphthyl acetate) is reported to be
researchers established FRET systems using them,3d,e such as 0.019 Æ 0.001 U mLÀ1.9 Up to now, several fluorescent sensors for
quantum dots, and silica and polymer particles. However, this detecting CaE have been reported,10 but most of them focused
architecture is sometimes problematic for obtaining ideal on imaging it in cells. While, establishing a reliable fluorescent
ratiometric sensors, because most fluorophores usually display system for detecting carboxylesterase in serum is of great impor-
aggregation-caused quenching (ACQ) properties,4 hence it is tance in terms of clinical applications.
hard to achieve strong donor emission and high energy transfer
Herein we designed and fabricated a FRET-based ratiometric
fluorescent system as CaE assay, by taking advantage of the
efficient energy transfer between tetraphenylethene derivative
(TPE-N+) nanoaggregate (AIE dots or TPE-N+ dots) and fluores-
cein. In this assay system, AIE dots serve as donors in the FRET
system. As a proof of concept, we choose fluorescein diacetate
(FDA) as the substrate for esterase activity study. The schematic
illustration for the ratiometric fluorescent detection is shown in
Fig. 1. The FDA substrate is neutral and nonfluorescent. In the
College of Materials Science and Engineering, State Key Laboratory of Luminescent
Materials and Devices, South China University of Technology, Guangzhou 510640,
P. R. China. E-mail: mcfzeng@scut.edu.cn, shzhwu@scut.edu.cn;
Fax: +86 20 22236363; Tel: +86 20 22236262
† Electronic supplementary information (ESI) available: Experimental section,
schemes, 1H NMR spectra, mass spectra, AIE behaviour, energy transfer effi-
ciency, quantum yield, absorption and emission spectra, the detection limit,
selectivity and tables. See DOI: 10.1039/c5cc04771d
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containing different water fractions. It reaches 8.75% in the
95% water/DMSO mixture and 9.65% in the powder state,
which is B30-fold higher than that of its DMSO solution
(molecularly dissolved state), as shown in Fig. S11 and Table S1
(ESI†). In contrast, the quantum yield of dansyl-N-butylamine
(DNS-NA, a typical non-AIE fluorophore (ACQ fluorophore) exhibits
similar spectral properties as the AIE dots) dramatically decreases
in the 90% water/DMSO mixture (Table S1, ESI†). In addition, the
photostability of the AIE dots was also investigated and compared
it with that of non-AIE fluorophores; the results are shown in
Fig. S12 (ESI†). It can be seen that upon continuous illumination
at 365 nm (15 W handheld UV lamp) for 2 h, only 4.4%
fluorescence intensity loss for AIE dots has been recorded, while
the other two non-AIE fluorophores whose spectral properties are
similar to that of the AIE dots show obvious photobleaching
phenomenon (Fig. S12, ESI†). This observation indicates that
the AIE dots have good photostability and can serve as promising
Fig. 1 Schematic illustration for the assay system and its ratiometric
fluorescence response to CaE. Photographs were taken under hand-
held UV light (365 nm).
absence of CaE, there is no electrostatic interaction between the energy donors in nanoparticle-based FRET systems.
TPE-N+ dots and the non-fluorescent FDA. No FRET occurs and
To investigate the fluorescence response of the assay system
only the TPE-N+ dots’ emission is detectable upon excitation towards CaE, we measured the fluorescence spectra of the sensing
at 355 nm. After addition of CaE, FDA undergoes catalytic system (TPE-N+ 30 mM and FDA 0.3 mM in PBS containing 15%
hydrolysis to yield fluorescent fluorescein molecules, which have DMSO) in the absence or presence of CaE. The fluorescence
two negative charges at pH = 7.4.10a Electrostatic attraction spectra of the system were periodically recorded during the
between the cationic TPE-N+ dots and anionic fluorescein mole- incubation of the assay system with CaE at 37 1C, and the results
cules brings them into close proximity so that the FRET process are shown in Fig. 2a–c. In the absence of CaE, the assay system
readily occurs, and the ratio of the two fluorescence intensities shows intense blue emission at 460 nm from TPE-N+ dots. Upon
serves as the sensing signal for the enzyme activity/level. The addition of CaE and with increased CaE incubation time, the blue
assay herein is applied to the detection of CaE activity/level in emission intensity of TPE-N+ dots at 460 nm gradually decreases
such biological fluid as human serum.
over the incubating time from 0 to 30 min. Meanwhile, the
The positively charged TPE derivative (TPE-N+) and fluores- emission of fluorescein at 520 nm shows a gradual increase.
cein diacetate were synthesized according to the synthetic routes After 30 min, the fluorescence intensity of the system and the
as shown in Scheme S1 (ESI†). And these compounds were fluorescence intensity ratio I520/I460 level off, as shown in Fig. 2b.
characterized by 1H NMR and mass spectrometry (Fig. S1–S7, In the meantime, we also studied the size change of TPE-N+ dots
ESI†). Afterwards, the TPE-N+ dots (AIE dots) were prepared by before and after CaE incubation using TEM and DLS. The dots
dissolving TPE-N+ in small amount of DMSO, and then diluting have an average diameter of about 40 nm before CaE addition.
it with PBS (1 mM, pH = 7.4) under stirring; TPE-N+ nano-
aggregates readily formed due to hydrophobic interaction.
Then fluorescein diacetate (FDA) solution was added to TPE-
N+ dots suspension, thus forming the sensing system. The AIE
behavior of TPE-N+ is shown in Fig. S8 (ESI†). In the DMSO/
water mixture solution, as the water fraction is increased, the
emission of TPE-N+ enhances.
Before investigating the fluorescence response of the sensing
system towards CaE, we first studied the spectral properties of
TPE-N+ dots and fluorescein. The normalized absorption and
emission spectra of TPE-N+ dots and fluorescein are shown in
Fig. S9 (ESI†). It can be seen that TPE-N+ has absorption and
emission at 300–360 nm and 430–480 nm, respectively, whereas
fluorescein exhibits absorption and emission at 460–500 nm
and 510–560 nm, respectively. Hence, there is a good spectral
overlap between the emission of TPE-N+ dots and the absorption
of fluorescein, which should favor the energy transfer between
Fig. 2 Time-dependent emission spectra (a) and fluorescence intensity as
a function of time (b) in the presence of CaE 20 U LÀ1; the fluorescence
them. In addition, upon varying the amount of fluorescein
intensity ratio as a function of time (c) and fluorescence spectra in the
added into the TPE-N+ dots dispersions, the energy transfer
presence of different CaE levels at 30 min upon addition of CaE (d) of the
sensing system (TPE-N+ 30 mM, FDA 0.3 mM). Inset in (d): fluorescence
intensity ratio as a function of CaE level. Excitation wavelength: 355 nm.
efficiency of the system can be adjusted (Fig. S10, ESI†). Also,
we measured the quantum yields of TPE-N+ in the solutions
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herein using the calibration curve (Fig. 3D, inset) as the standard,
and the endogenous CaE level for the undiluted serum sample is
then calculated as 17.2 U LÀ1. Furthermore, the assay system was
also compared with
a commercially available colorimetric
method, by which the naphthyl acetate hydrolyzed product was
measured at 321 nm (e321 = 2200 MÀ1 cmÀ1).9 The endogenous
esterase level in serum determined using the colorimetric method
is 16.4 U LÀ1, which is close to that determined by our system.
For the colorimetric assay, the detection process requires
complex multiple procedures, whereas for the assay system
herein, the detection process is quite convenient and could
serve as a one-step straightforward assay.
In addition, the recovery of the added known amounts of
CaE into the serum samples is in general more than 94% by the
assay system, which suggests the accuracy and reliability of the
present method for esterase determination. Furthermore, the
precision of the assay system was also investigated (Table 1), which
Fig. 3 HR-TEM images of the assay systems before (A) and after (B) the
addition of 20 U LÀ1 CaE (placed on copper grids). Scale bar: 50 nm. Size
distribution of the assay system determined by dynamic light scattering
(DLS) before (C) and after (D) the addition of 20 U LÀ1 CaE.
After the addition of CaE and with the incubation of 30 min, the was determined using the relative standard deviation. It is obvious
size of the nanoaggregates becomes a little larger, about 50 nm as that the assay system herein displays quite good precision.
shown in Fig. 3. The size changes determined by DLS (B45 nm
Compared with other reported fluorescent probes for car-
and 70 nm) are slightly larger than those determined using TEM. boxylesterase (as shown in Table S2, ESI†), the probe system
Also, we measured the fluorescence spectra of the sensing herein can realize carboxylesterase detection in the fluores-
system in the absence or presence the different concentrations cence ratiometric mode, while current fluorescent probes for
of CaE, as shown in Fig. 2d. It is clear that, as the enzyme level carboxylesterase mostly function in turn-on mode; further-
is increased, the emission of the TPE-N+ dots decreases, while more, the probe system can detect carboxylesterase in human
that of the fluorescein enhances; therefore, this sensing system serum samples and could serve as a convenient straightforward
can detect the enzyme level in a ratiometric way, and the one-step fluorescence assay for carboxylesterase.
detection limit is determined as 0.26 U LÀ1 (Fig. S13, ESI†).
In summary, we have successfully developed a novel ratio-
Moreover, the fluorescence intensity of the assay system metric fluorescence assay system based on AIE dots as the FRET
with AEBSF (a potent CaE inhibitor, 1 mM) in the presence of donors, which can avoid the limitations of the donors consisting
CaE (20 U LÀ1) was measured (Fig. S14, ESI†). The enzyme of aggregation-caused quenching fluorophores in nanoparticle-
inhibition by AEBSF demonstrates that the fluorescence based FRET systems. The obvious advantages of employing AIE
change is indeed induced by CaE. To study the selectivity of dots include: first, they exhibit strong emission in the aggregated
the system, various potential interfering species were examined state, which is beneficial for the nanoparticle-based systems;
in parallel under the same conditions. As shown in Fig. S15, second, they have better photostability, which is good for
ESI,† the system shows a high selectivity towards CaE over the applications for a longer period. Also, the assay system features
other species tested.
sensitive and selective detection of carboxylesterase in aqueous
To evaluate the efficacy of this assay system in real samples, media and biological milieus. This strategy may provide a new
the system was applied to measure the level of carboxylesterase and effective approach for establishing new FRET systems and
in the serum. The serum samples were diluted 50-fold for the developing other enzyme assays.
measurements, and the determined carboxylesterase levels are
We gratefully acknowledge the financial support from the
listed in Table 1. The endogenous (originally existing) CaE in National Key Basic Research Program of China (Project no.
the diluted serum sample was determined by the assay system 2013CB834702), NSFC (21474031, 21174040 and 21025415) and
Table 1 Determination of carboxylesterase (CaE) in serum (50-fold diluted)
Determined endogenous
CaE (U LÀ1) by colorimetry
Determined endogenous
CaE (U LÀ1) by this probe
Added CaE
(U LÀ1
Combined
CaE (U LÀ1
Measured (U LÀ1
by this probec
)
)
)
Recovery (%)
0.328 Æ 0.012a
0.344 Æ 0.007b
104.9
94.6
100.2
101.5
98.8
6.000
10.000
20.000
30.000
6.344
10.344
20.344
30.344
6.001 Æ 0.010
10.365 Æ 0.005
20.649 Æ 0.014
29.980 Æ 0.008
a
The carboxylesterase level in the 50-fold diluted serum sample is 0.328 U LÀ1, which means the endogenous carboxylesterase concentration in
undiluted serum is ca. 0.0164 U mLÀ1, determined by the colormetric method. The carboxylesterase level in the 50-fold diluted serum sample is
b
0.344 U LÀ1, which means the endogenous carboxylesterase concentration in undiluted serum is ca. 0.0172 U mLÀ1, determined by the assay
c
system herein. The data are summarized as mean Æ standard deviation (SD).
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