A simple design of fluorescent probes for indirect detection of β-lactamase based on AIE and ESIPT processes

Article abstract of DOI:10.1039/c8tb00414e

A novel fluorescent probe DNBS-CSA is developed for light-up detection of β-lactamase. The probe design is based on an indirect detection approach with three step reactions. β-Lactamase can react with the lactam of its substrate (cefazolin sodium) to prod

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A simple design of fluorescent probe for indirect detection of β-
lactamase based on AIE and ESIPT processes
Received 00th January 20xx,
Accepted 00th January 20xx
Lu Peng, Lu Xiao, Yiwen Ding, Yu Xiang and Aijun Tong*
DOI: 10.1039/x0xx00000x
A novel fluorescent probe DNBS-CSA is developed for light-up detection of β-lactamase. The probe design is based on an
indirect detection approach with three step reactions. β-Lactamase can react with the lactam of its substrate (cefazolin
sodium) to produce a secondary amine, initiating a spontaneous elimination reaction and affording a thiol compound. The
thiol could further react with the sulfonate group of DNBS-CSA, releasing the salicylaldehyde azine derivative (CSA) with
both aggregation induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) characteristics.
Previously reported β-lactamase probes require covalent linkage of the substrate β-lactam ring part to probe, which makes
the probe synthesis difficult due to the complicated structure of β-lactam ring. On the contrary, modification of β-lactam
ring is no longer necessary for DNBS-CSA according to our indirect detection approach. The linear range of fluorescent
quantification for β-lactamase is 0-10 mU/mL in aqueous solution. Moreover, owing to the AIE property of CSA, detection
of β-lactamase with DNBS-CSA on test papers was also achieved.
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synthesis. Compared with the substrates of other enzymes, the
substrate (β-lactam ring) of β-lactamase has a much more
complicated structure. The previously reported β-lactamase
probe all adopted a direct detection approach, requiring
tedious modification of substrate (β-lactam ring) to probe.
Thus, it is still very challenging to develop novel detection
approach for β-lactamase, which should not only match up
with the requirement of sensitivity, detection time and ease of
real sample analysis, but also avoiding the complex
modification of β-lactam ring.
Moreover, the reported fluorescent probes for β-lactamase
are designed with fluorophores suffering from aggregation
caused quenching (ACQ),32, 33 such as indolium and fluorescein,
which limited their application when used in low β-lactamase
content detection since fluorescence signal could not be
elevated by increasing probe concentration. Besides, due to
the ACQ effect, it is hard to apply these probes for β-lactamase
detection in solid state, such as on test papers, despite that
paper-based sensors34, 35 could find potential application in
point-of-care detection due to their portable, inexpensive,
disposable and low sample volume requirement. Therefore,
developing β-lactamase probes that allows detection in solid
state without ACQ effect is still needed.
Introduction
β-lactamase is an important bacterial enzyme that can cleave
the amide group of β-lactam antibiotics1-3 (penicillins,
cephalosporins, etc.) with high catalytic efficiency, which
makes the β-lactamase producing bacteria, such as Gram-
negative bacterium4 and Staphylococcus5, resistant to β-
lactam antibiotics. Thus, β-lactamase is a significant biomarker
for identification of bacterial pathogens that are resistant to β-
lactam antibiotics.6 In addition, there are reports7, 8 on illegal
addition of β-lactamase into milk to conceal the antibiotics
residue by unethical manufacturers, in order to reuse the
contaminated milk illegally. Therefore, the sensitive detection
of β-lactamase is of great importance.
Fluorescent probes for various analytes have attracted much
attention owing to the simplicity, low cost, good sensitivity,
and capability of real-time detection, which enable effective
in-vitro and in-vivo studies.9-22 So far, many fluorescent probes
for enzyme activity detection have been reported, such as
24
esterase23,
and alkaline phosphatase probes25 based on
hydrolisis of ester group, pantetheinase probe based on
cleavage of pantetheine,26 nitroreductase probe based on
reduction of nitro group.27 However, only limited probes for β-
Fluorophores with aggregation induced emission (AIE)36-43
are a series of molecules emitting strong fluorescence in their
aggregate or solid states. Up to now, many AIE fluorophores
based on hydrocarbons, heteroatoms or organometallics have
been constructed as fluorescent materials for biosensing
applications.40, 44-46 Previously, we have developed a class of
AIE fluorophores based on salicylaldehyde azines (SA)47 with
lactamase have been developed.3,
The scarcity of β-
28-31
lactamase probe is mainly due to the difficulty of probe
Department of Chemistry, Beijing Key Laboratory for Analytical Methods and
Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical
Biology, Tsinghua University, Beijing 100084, PR China.
*E-mail: tongaj@mail.tsinghua.edu.cn
Electronic Supplementary Information (ESI) available: [characterization of DNBS-
49
excited state intramolecular proton transfer (ESIPT)48,
mechanism. The hydroxyl groups in these molecules are
CSA and supplementary figures]. See DOI: 10.1039/x0xx00000x
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Synthesis of compound DNBS-CSA and CSA
responsible for ESIPT and essential for their AIE characteristics
in aggregate and solid states. Therefore, a universal SA-based
AIE probe design strategy were proposed through protection
and deprotection of the hydroxyl groups. Based on this
strategy, we have developed several fluorescent probes for the
detection of thiol,50 beta-galactosidase51 and esterase,52
demonstrating the versatility of the platform.
View Article Online
DOI: 10.1039/C8TB00414E
Compound DNBS-CSA and CSA were prepared according to our
previously reported procedure.50 The molecular structures were
confirmed to be right by the NMR and MS spectroscopy (details can
be found in Supplementary Information).
Analytical procedures
Herein, we report a simple design of fluorescent probe for
indirect detection of β-lactamase based on AIE and ESIPT processes.
The indirect detection approach incorporates three-step reactions
(Scheme 1): firstly, a cephalosporin substrate 1 of β-lactamase was
introduced into the detection system to cleave the β-lactam ring
and release a secondary amino group; secondly, the amino group
further triggers spontaneous elimination reaction to afford an
intermediate product 2 containing thiol; and thirdly, the thiol of 2
could further react with the sulfonate group of probe DNBS-CSA to
release the essential hydroxyl group, recovering the AIE
fluorescence. Thus, the activity of β-lactamase could be detected
indirectly by our previously reported thiol probe DNBS-CSA50. This
indirect detection approach does not need tedious modification of
β-lactam to probe, while strong yellow AIE fluorescence was
observed in the presence of β-lactamase, allowing the light-up
detection of β-lactamase activity both in solution and on test
papers.
The stock solution (500 μM) of DNBS-CSA was prepared in DMSO.
Stock solutions of cefazolin sodium salt (8.0 mM), β-lactamase (10.0
U/mL) and other analytes (CaCl2, Mg(NO3)2, Zn(NO3)2, Fe(NO3)3,
Cu(NO3)2, pepsin, lysozyme, vitamin C, alanine, BSA, lactose, casein,
and esterase) were prepared in PBS buffer (10 mM, pH 7.4),
respectively. In a typical detection procedure, 150 μL DNBS-CSA
stock solution and 300 μL cefazolin stock solution were added into a
test tube. Subsequently, a proper amount of β-lactamase stock
solution was added, making the total volume of sample solution to
be 500 μL. After reaction at 37 ℃ for different time, the
fluorescence spectra were recorded.
For the β-lactamase sensing in real samples, test paper and milk
sample solution were prepared, respectively. By dropping 5 μL
solution of DNBS-CSA in THF (10 mM) on the filter paper strip and
underwent evaporation of THF for 3 min, test paper was fabricated.
In a typical procedure, sample solution containing 10% milk was
prepared by adding 600 μL cefazolin stock solution (8.0 mM), 100
μL milk, different volume of PBS buffer and β-lactamase stock
solution (10.0 U/mL or 0.1 U/mL) into a test tube, making the total
volume of sample solution as 1000 μL. The test paper was
subsequently dipped into the sample solution. After incubation at
80 ℃ for 20 min, the test paper was taken out and dried for 30 min.
Photographs of test paper was taken under a UV lamp and the
Image J software was used to read the intensity of fluorescent spot
on test paper. The β-lactamase concentration in sample solution
was then calculated through the calibration curve.
Results and discussion
Probe design
Scheme 1 An indirect approach for fluorescent light-up detection of β-
lactamase by using probe DNBS-CSA.
As shown in Scheme 1, DNBS-CSA was synthesized for indirect
detection of β-lactamase. In the structure of DNBS-CSA, one
hydroxyl group was blocked by sulfonate and thus the ESIPT process
was restricted. Besides, the nitro groups on benzene sulfonate
could serve as effective quencher. Therefore, DNBS-CSA was
initially non-fluorescent. After the addition of β-lactamase, it
cleaved the β-lactam ring of the cefazolin substrate and created a
free secondary amino group, which triggered spontaneous
elimination of the leaving group previously attached to the 3’
position1 (Scheme 1a ). Thus, an intermediate product 2 containing
thiol was afforded, which further cleaved the sulfonate group of
DNBS-CSA, removing the 2, 4-dinitrobenzenesulfonate group and
recovering the hydroxyl group, yielding CSA. While in CSA, two
essential hydroxyl groups were present that enabled the ESIPT and
the AIE fluorescence. In this way, an indirect fluorescent light-up
detection method for β-lactamase was established.
Experimental
Materials and instruments
β-Lactamase from Enterobacter cloacae and other chemicals were
purchased from Sigma-Aldrich (St. Louis, USA) and used without
further purification. Plain milk was used as real sample without
further pretreatment.
All fluorescence spectra were determined on a fluorescence
spectrometer (JASCO FP 8600, Japan). All NMR spectra were
recorded using a NMR spectrometer (JEOL JNM-ECA300, Japan).
The mass spectra were obtained on an ion trap time-of-fight mass
spectrometry (Shimadzu MS-IT-TOF, Japan). The pH measurement
was performed with a pH meter(Sartorius PB-10 basic, Germany).
The AIE characteristic of CSA
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fluorescence intensity (I558) change over concentration of 2. (b) Fluorescence
We firstly studied the effect of water volume fraction (fw= 0-99 vol%)
on the fluorescence of CSA in water/DMSO co-solvents, and the
results are shown in Fig. 1. CSA (150 μM) is soluble in DMSO but is
not in water. In a good solvent DMSO, CSA disperses well with no
fluorescence. However, in a poor solvent (fw= 99 vol%), an intense
fluorescence band at 558 nm can be observed. The AIE effect of
CSA occurs when the water volume fraction in the co-solvent is
more than 30% and fluorescence intensity could be further
enhanced with higher water volume fractions, which correlates well
with the increased aggregates formation in poorer solvents. The AIE
fluorescence is originated from restriction of free intramolecular
rotation assisted by intramolecular hydrogen bonds of CSA in the
aggregate state and from the excited state intramolecular proton
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intensity (I558) change over time.
DOI: 10.1039/C8TB00414E
Then, the ability of DNBS-CSA as a sensor for β-lactamase
detection was examined. As can be seen in Fig. 3, the addition of β-
lactamase induced a gradual fluorescence intensity enhancement at
558 nm. In order to enable fast detection, we recorded the
fluorescence intensity (I558) change upon addition of β-lactamase
for 8 min. It was found that the increasement rate of I558 within 5-8
min had
concentration ranging from 0 to 10 mU/mL. The detection limit was
calculated as 0.5 mU/mL based on the definition by IUPAC (CDL
a good linear relationship with the β-lactamase
=
3Sb/m) from 10 blank samples. This analytical performance could
match up with the previously reported β-lactamase probes based
on direct detection approach (see comparison of analytical
performance in Table S1 in the ESI.†).
transfer (ESIPT)48-50
.
Fig. 3 Fluorescence intensity (I558) change of DNBS-CSA (150 μM) in the
presence of various concentrations of β-lactamase (0-100 mU/mL) in PBS
solution containing cefazolin sodium (4.8 mM). (a) Time dependence; (b) β-
lactamase concentration dependence, inset: calibration curve of I558
increasement rate within 5-8 min versus β-lactamase concentrations. The
I588 increasement rate is calculated by the formula (I-I0)/t, where I and I0 are
the I558 value at 5 min (300 s) and 8 min (480 s), respectively, while t equals
to 180 s.
Fig. 1 (a) Effect of water volume fraction on the (a) fluorescence spectra and
(b) intensity (I558) of CSA (150 μM）in water/DMSO containing 10 mM PBS
buffer at pH 7.4. Excitation was set at 383 nm. (c) The corresponding
photographs of CSA with a UV lamp at 365 nm.
Study of β-lactamase activities in solution
It has been reported by Tsien’s group1 and others2 that β-
lactam can be cleaved by β-lactamase to afford a secondary
amino group. As for cephalosporin, this secondary amino group,
could further trigger spontaneous elimination of any leaving
group previously attached to the 3’ position. On the other hand,
the reaction between sulfonate and thiol to release hydroxyl has
been reported by Maeda’s group53 and others50, 54. To verify the
proposed mechanism in Scheme 1, we conducted three
confirmation experiments, respectively: firstly, the formation of 2
by cefazolin and β-lactamase was confirmed (Fig. S1 in the ESI.†);
secondly, the release of CSA by probe DNBS-CSA and 2 was
confirmed (Fig. S2 in the ESI.†). And finally, for the mixture of
cefazolin, β-lactamase and DNBS-CSA, the result of mass spectrum
clearly suggested the formation of CSA (Fig. S3 in the ESI.†),
supporting the hypothesis in our design (Scheme 1).
The fluorescence of DNBS-CSA in the absence and presence of the
thiol containing intermediate product 2, was first studied to prove
our design principle above (Scheme 1). As shown in Fig. 2a, DNBS-
CSA (150 μM) displayed no fluorescence in PBS buffer (10 mM, pH
7.4), suggesting successful blocking effect of hydroxyl by sulfonate
group and quenching effect by nitro groups. Upon addition of 2, the
fluorescence of DNBS-CSA enhanced gradually until strong yellow
fluorescence (558 nm) was observed. The time-dependent
fluorescence intensity change of DNBS-CSA was recorded without
and with the addition of 2 (Fig. 2b), showing that the fluorescent
light-up response only occurs in the presence of 2.
Study of β-lactamase activities on test papers
Because of the AIE characteristic of CSA, we further investigated
the possibility of using DNBS-CSA as a portable test paper sensor
for the detection of β-lactamase. Test papers were fabricated by
dropping 5 μL solution of DNBS-CSA in THF (10mM) on the filter
paper strips. After evaporation of THF, test papers were dipped into
the sample solution containing cefazolin (4.8 mM) and different
concentration of β-lactamase (0-7.0 mU/mL) for 20 min. Owing
to the AIE characteristic of CSA, the fluorescence change of
Fig. 2 (a) Fluorescence spectra of DNBS-CSA (150 μM) in the presence of
various concentrations of 2 (0-7.5 mM) for 30 min in PBS buffer solution (10
mM, pH 7.4, 37 ℃). Insets from left to right: photographs of DNBS-CSA (150
μM) without or with 2 (7.5 mM) under UV light (365 nm); the corresponding
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DNBS-CSA towards β-lactamase was clearly observed by naked eyes
(Fig. 4a). Image processing software Image J was used to read the
intensity of fluorescent spots (Fig. S4 in the ESI.†). As can been
seen in Fig. 4c, a good linearity could be found in the β-lactamase
concentration range of 0-2.0 mU/mL.
recoveries and standard deviation values, suggestiVniegw DArNticBleSO-CnlSinAe
DOI: 10.1039/C8TB00414E
could find potential use for β-lactamase detection in milk sample.
Conclusions
In summary, we have proposed a simple design of fluorescent
probe for indirect detetction of β-lactamase activity based on
AIE and ESIPT processes. Upon the addition of β-lactamase,
the indirect detection approach with three-step reactions
could be initiated. The intermdiate thiol containing product of
cefazolin and β-lactamase could furhter react with probe
DNBS-CSA, thus recovering ESIPT and strong fluorescence in
the aggregate and solid states. Unlike other β-lactamase
probes require complicated covalent modification of substrate
(β-lactam ring) to probe, DNBS-CSA responds to β-lactamase in
an indirect approach, making the modification of β-lactam ring
no longer necessary. Moreover, thanks to the AIE
characteristic of CSA, the light-up detection of β-lactamase
was achieved both in aqueous solution and on test papers. For
enzymes which are difficult to develop “direct approach”
probes due to the complicated structures of substrates, this
“indirect approach” probe design strategy provides
Fig. 4 (a) Photographs of test papers under a UV lamp (365 nm) for the
detection of β-lactamase at various concentrations (0-7.0 mU/mL) in PBS
solution containing cefazolin sodium (4.8 mM). (b) The corresponding
fluorescence intensity of spots read by Image J software versus the
concentrations of β-lactamase. (c) Calibration curve for β-lactamase opportunities by detecting a simpler intermediate product.
detection.
The selectivity of DNBS-CSA towards β-lactamase was also
studied (Fig. S5 in the ESI.†). Upon the addition of β-lactamase,
the fluorescence intensity of DNBS-CSA was enhanced. However,
Conflicts of interest
There are no conflicts to declare.
almost no fluorescence intensity change was observed for some
commonly used inorganic salts or biomolecules, such as Mg(NO3)2,
CaCl2, vitamin C, lysozyme and esterase. Thus, DNBS-CSA shows
good selectivity to β-lactamase.
Acknowledgements
We are very grateful for the financial support from the
National Natural Science Foundation of China (Nos.
21775084, 21390410 and 21621003) and China Postdoctoral
Science Foundation (2016M601000).
Table 1. Determination of β-lactamase in sample solution containing 10%
milk.
Notes and references
Sample
β-lactamase
Added
Found
Recovery
(%)
R. S. D.
(%)
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A fluorescent probe with both AIE and ESIPT characteristics has been developed for βꢀlactamase based on an indirect approach.
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