An HBT-based fluorescent probe for nitroreductase determination and its application in: Escherichia coli cell imaging

Article abstract of DOI:10.1039/d0nj03286g

A novel HBT-based fluorescent probe HBTPN was reasonably designed for detecting nitroreductase by merging 4-nitrobenzyl as a sensing moiety to HBT-based dye HBTPP. Experiments showed that probe HBTPN displayed a selective and sensitive "turn-on"response t

Full text of DOI:10.1039/d0nj03286g

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An HBT-based fluorescent probe for nitroreductase determination
and its application in Escherichia coli cell imaging
Received 00th January 20xx,
Accepted 00th January 20xx
Quanzhi Yang,a b Yujie Wen,a b Aiguo Zhong,c Jian Xua and Shijun Shao*a
DOI: 10.1039/x0xx00000x
A novel HBT-based fluorescent probe HBTPN was reasonably proton transfer), and the emission spectrum could be
designed for detecting nitroreductase by merging 4-nitrobenzyl as redshifted to long-wavelength region via extending the π-
11, 12
a sensing moiety to HBT-based dye HBTPP. Experiments showed conjugation system with electron-accepting unit.10,
that probe HBTPN displayed selective and sensitive “turn-on” Exemplary HBT-based fluorophores (HBTP and HBTPP) have
response to nitroreductase and has been applied for sensing been developed by introducing cationic picoline salt as the
nitroreductase in living Escherichia coli.
electron acceptors, which were reported exhibiting large
Stokes shift and long wavelength emission because of ESIPT
coupled ICT (intramolecular charge transfer) mechanism
(Scheme 1).13 Through masking their phenolic hydroxyl with
detecting group and thus blocking the ESIPT process, various
probes were developed for monitoring analytes including
hydrogen sulfide, hypochlorite, palladium, hydrogen peroxide,
alkaline phosphatase and nitroreductase, etc.13, 14
The flavin-containing nitroreductase (NTR), which was found
overexpressed in Enterobacter cloacae and hypoxic human
tumours, could catalyse the reduction of nitroaromatic
compounds to the corresponding hydroxylamines or amines
with cofactor NADH.1 This enzyme has been utilized in
bioremediation of 2,4,6-trinitro toluene (TNT) and other
nitroaromatic pollutants in the past years.2 Nowadays, much
attentions were paid to the development of NTR sensors, and
most of them were focused on their applications in assessing
hypoxic status of solid tumours.3 In view of the high-
expression of NTR in Escherichia coli (E. coli), we speculated
that this enzyme was promising to be designed as a potential
reporter to monitoring infections and food contamination
caused by E. coli.4, 5, 6
Scheme 1. Design of fluorescent probes based on HBTPP scaffold.
Among all the NTR assays, fluorescence technology was
desirable due to its high spatiotemporal resolution and
nondamaging detection, and remarkable progress has been
made in the development of NTR fluorescent probes recently.7,
Inspired by those findings, a novel fluorescent probe (HBTPN
for NTR was designed by merging 4-nitrobenzyl group as
responsive unit to dye HBTPP (Scheme 1). Different from the
previous strategy of masking the hydroxyl group, the detecting
group of probe HBTPN was merged with the pyridinium unit
without blocking the ESIPT process.15 We speculate that 4-
nitrobenzyl moiety functioned not only as the NTR detecting
unit, but also as alkylating agent which provide the probe
characteristic of long wavelength emission. Furthermore, upon
addition of NTR, nitroaromatic group might be reduced to
hydroxylamines or amines, which would weaken the electron-
withdrawing effect of the pyridinium moiety as well as the ICT
process and the possible photoinduced electron transfer (PET)
process from HBTPP moiety to the nitrobenzyl group, result in
changes of fluorescence signal.7, 16
)
8
However, most of the fluorescent probes suffered from
drawback of short wavelength emission or small Stokes shift
(Table S1 in ESI†), which may lead to disadvantages such as
poor penetration, severe photodamage and crosstalk between
the excitation and emission spectra.9 Hence, the development
of novel probes for NTR with enlarged Stokes shift as well as
long wavelength emission was becoming increasingly
important. Recently, HBT (2-hydroxyphenyl-substituted
benzothiazole) and its analogues have been frequently used in
developing fluorophores with large Stokes shifts due to their
intrinsic mechanism of ESIPT (excited-state intramolecular
Probe HBTPN was synthesized following the procedures
reported previously.10, 15 To investigate the sensing mechanism,
a.CAS Key Laboratory of Chemistry of Northwestern Plant Resources and
Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute
of Chemical Physics, Chinese Academy of Sciences, Lanzhou, Gansu
730000, P. R. China.
b.University of Chinese Academy of Sciences, Beijing 100049, P. R. China.
c. Department of Chemistry, Taizhou College, Taizhou 318000, P. R. China
†Electronic Supplementary
DOI: 10.1039/x0xx00000x
Information
(ESI)
available:
See
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a novel HBT-based fluorophore HBT-2Py (Scheme 2) was confirmed by HPLC (Fig. S7 in ESI†). After being inVciuewbaArtteicdle Ownilitnhe
synthesized for comparison.17 Details of synthesis route and nitroreductase, a decrease in the peak at 6.00 min on behalf of
DOI: 10.1039/D0NJ03286G
characterization were given in the Supporting Information (SI). HBTPN was observed, together with the emergence of a new
The optical properties of HBTPN were then measured in peak at 11.69 min ascribed to HBT-2Py and an uncharted peak
different conditions. Complex absorption spectra were at 3.56 min. The LC-MS spectrum of the uncharted peak (Fig.
observed in HBTPN with strong absorption bands around 300 S6B in ESI†) provided a signal of hydroxylamine compound
and 410 nm in solvents (Fig. S1A in ESI†), and the absorption (m/z=466.0), suggesting that this peak could be assigned to
bands (in DMSO and CH3OH) after 475 nm were reasonably hydroxylamine compound. Further experiments demonstrated
ascribed to partial deprotonation of phenol unit (Fig. S2A in that fluorescence signal of HBT-2Py would be partly inhibited
ESI†).17, 18 In the fluorescence spectra, due to the mechanism by the coexistence of HBTPN and the addition of part of HBT-
of ESIPT coupled ICT process, longer emission wavelength 2Py could hardly change the emission curve of reaction
(λem≥638 nm) and larger Stokes shift (Δλ≥232 nm) were mixture (Fig. S8 in ESI†). Based on these experiments, a
obtained in probe HBTPN (Table S2 and Fig. S3 in ESI†). The plausible mechanism was proposed that probe HBTPN
fluorescent intensity decreased gradually from nonpolar underwent NTR mediated nitro reduction to yield
solvent CH2Cl2 to polar solvent (CH3CN, CH3OH and DMSO) hydroxylamine compound (Scheme 2).21 Inevitably, this
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with the emission band becoming red-shifted (Fig. S1B in ESI†), compound might partly decompose into HBT-2Py
which was in conformity with the report that the fluorescence
property of HBT derivatives were sensitive to solvents,12, 19 and
notably, extremely weak fluorescence signal was detected in
strong polar/protic water with a low quantum yield of
Φ=0.0026.
.
Subsequently, the fluorescence responses of probe to NTR
were evaluated in Tris-HCl (pH=7.4, 50 mM) with NADH (25 μM)
Scheme 2 Probe HBTPN and its reaction mechanism with NTR.
as electron donor. The detecting solution of HBTPN (5 μM)
displayed negligible emission signal in red/NIR region (Fig. 1).
However, upon the addition of NTR, 22-fold increasement of
fluorescence intensity at 633 nm was obtained, which
validated HBTPN as a potential “turn-on” probe.
Density functional theory (DFT) calculations were conducted
to further investigate this response mechanism (Table S3 in
ESI†).16 As demonstrate in natural transition orbitals (NTOs),
the lowest energy absorption band resides on the HBTPP
moiety with the intramolecular charge transferred from HBT
unit to pyridinium part for both probe and hydroxylamine
compound. On the singlet emitting states the π electrons of
HBTPP was delocalized over the electron-withdrawing nitro
aromatic moiety in probe, while no electron transfer was
observed for hydroxylamine compound. This confirms that the
nitro group induced PET process, result in the weak
fluorescence of probe in water, and that blocking of the PET
effect lead to the strong fluorescence signal in hydroxylamine
compound.
The fluorescence kinetic responses of probe HBTPN reacting
with NTR were then investigated, which demonstrated that
fluorescence spectra exhibited obvious changes along with
reaction process (Fig. S9 in ESI†). Fluorescence intensity at 633
nm increased rapidly and reached to the maximum in about 10
min with 4-17 fold enhancement at various concentration,
while hardly any change was observed without NTR during the
same period. Moreover, negligible fluorescence intensity
change was observed after being continuously illuminated for
75 minutes, and the absorption and emission spectra changed
slightly at a temperature range of 0-60°C, suggesting the
photostability of probe HBTPN (Fig. S4 and S10 in ESI†).
Fig. 1 Emission spectra of HBTPN, HBT-2Py (5 μM) and the reaction
system (HBTPN incubated with 1.5 μg/ mL NTR), λex=397 nm; Insert
picture: fluorescence photograph of reaction solution.
Notably, the emission band of reaction mixture was
different with that of HBT-2Py, which exhibited strong
fluorescence emission centred at 595 nm, indicating that
probe HBTPN may not undergo the usual mechanism of NTR-
catalysed reduction followed by rearrangement-elimination to
liberate HBT-2Py 20
To explore the sensing mechanism, ESI-
.
HRMS experiments were conducted and results indicated that
HBTPN converted into a molecular with ion peak at m/z
466.1566 (Fig. S6A in ESI†), which could be logically
characterized as the hydroxylamine compound reduced from
probe HBTPN (unfortunately, we failed to synthesize this
compound). Subsequently, these species were further
Subsequently, the selectivity of HBTPN to NTR was
measured. A variety of interfering species including inorganic
salts, reactive oxygen species, amino acids, biothiols, glucose
and vitamin C, failed to trigger fluorescence response whereas
only NTR could induce a remarkable increment of fluorescence
signal (Fig. S11 in ESI†). Moreover, the fluorescence intensity
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incubated with dicoumarol (NTR inhibitor) decreased obviously culture-based tests and PCR-based geneView sAertiqclue eOnnlcinee
and more dicoumarol would lead to a progressive decline. identification, which need complicateDeOqI:u1i0p.1m03e9n/Dt 0oNrJ03t2im86eG-
Those results demonstrate that HBTPN was catalysed by NTR costing and damaging pre-treatment, probe HBTPN should be
specifically in the detective system. Besides, effects of pH or promising to provide a facile, rapid and non-invasive method
temperature on reaction system (Fig. S12 in ESI†) revealed that to assess NTR activity in live E. coli.5, 6, 22 In this experiment, E.
HBTPN functioned well under physiological conditions (37 °C, coli were cultured in Luria–Bertani media, washed with Tris-
pH=7).
HCl buffer and then resuspended in Tris-HCl buffer without
Titration experiments were further carried out to test the lysis to test the absorbance and emission spectra. Incubated
performance of probe HBTPN for quantitatively detecting NTR. probe HBTPN with bacteria solution for 10 min could trigger
As depicted in Fig. 2, the fluorescence intensity at 633 nm obvious increase in fluorescence intensity compared with only
enhanced accompany by the increasing concentrations of NTR, the probe or E. coli sample (Fig. 3A), indicating that HBTPN was
and good linearity was exhibited in the range of 0.05–1.5 able to diffuse across the rigid cell wall and the plasma
µg/mL with detection limit determined as 2.9 ng/mL based on membrane, and reacted with NTR within bacterial cytoplasm.
3σ/k, suggesting HBTPN was able to detect NTR qualitatively However, the addition of dicoumarol (0.1 mM) to E. coli
and sensitively. The Michaelis constant (Km) and the maximum together with HBTPN led to significantly decrease in
of the initial reaction rate (Vmax) for the NTR-activated reaction fluorescence signal, confirmed the specificity of HBTPN to
were determined to be 25.00 μM and 0.014 μM/s, respectively, endogenous NTR in E. coli cell (Fig. S15 in ESI†). Encouraged by
which was comparable to the values reported previously (Fig. these results, the capacity of probe HBTPN for dynamic
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S13 in ESI†). 4, 7
detection the growth process of E. coli was investigated. As
shown in Fig. 3, the concentration of E. coli (which was
determined by the OD600 value) grew quickly at early stage and
then increased slowly, and notably the change curve of
fluorescence intensity fitted well with the growth status of E.
coli, validating the HBTPN as a potential E. coli growth
indicator.
Fig. 2 (A) Fluorescence response of probe (5 μM) with NTR at varied
concentrations; (B) Linear correlation between fluorescent intensities
and NTR concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8 ,1.0, 1.25, 1.5
μg/mL); Error bars stand for the mean standard deviation of three
results. λex 397 nm.
Docking studies were carried out to explore molecular
recognition procedure. In binding process, probe HBTPN
tended to approach the catalytic core of NTR by π-π stacking
force and the benzothiazole unit could bind to a hydrophobic
pocket by hydrophobic interactions (Fig. S14A in ESI†). The 4-
nitrobenzyl moiety access to a region delimited by residues
Ser12, Arg172, Arg11, Arg10, and seven hydrogen bonds were
constructed with these residues, which was in a similar binding
mode with the probe reported previously (Fig. S14B in ESI†).16
In the departure process, after being reduced to
hydroxylamine compound, only three hydrogen bonds were
formed (Fig. S14C in ESI†). The docking affinity was calculated
as 8.99 kcal/mol for hydroxylamine compound, which was
lower than that of probe HBTPN (10.59 kcal/mol). A low
docking value indicated that enzyme could dissociate from the
reduced compound easily and then prepare for the following
catalytic process. As a consequence, the binding and departure
process indicated hydrogen bond and docking affinity were
significant in the NTR-mediated reduce reaction, and that
probe HBTPN could be well catalysed by NTR.
Fig.
3 (A) Fluorescence intensity of different reaction solution;
bacterial concentration was OD600=0.12. (B) Time-based absorbance
change of E. coli in culture media. (C) Fluorescence intensity of HBTPN
incubated with E. coli after growing for different periods of time. Error
bars stand for the mean standard deviation of three results.
Fig. 4 Fluorescence images of E. coli cells incubated with HBTPN in the
absence and presence of dicoumarol.
The evaluation of NTR activity in living E. coli was further
conducted by confocal microscopy (Fig. 4). Strong fluorescence
signal was detected after incubated with HBTPN for 10 min
It is known that NTR was abundant in E. coli and the
assessment of this enzyme will be helpful to examine microbial
contamination.5 Compared with conventional methods like
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H. C. Huang, K. L. Wang, S. T. Huang, H. Y. Lin and C. M. Lin,
while negligible fluorescence was observed in E. coli cells alone.
Meanwhile, pre-treatment with dicoumarol before incubated
with HBTPN led to obvious decline in fluorescence intensity,
certified again that HBTPN could permeate the bacterial cell
wall and be specially activated by the intracellular
nitroreductase. These results demonstrated that probe HBTPN
was potential to act as a useful indictor to rapidly assess E. coli
caused infections.
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In summary, we have developed a novel HBT-based
fluorescent probe HBTPN for NTR by merging 4-nitrobenezyl
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,
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This study was financial supported from top priority program
of “One-Three-Five” Strategic Planning of Lanzhou Institute of
Chemical Physics and the National Natural Science Foundation
of China (21572239 and 21505145). Authors acknowledged Dr
Xiaoyan Yang for her help in E. coli culture experiment.
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HBTPN exhibited excellent performance such as rapid response time, large Stokes shift
highly selectivity and sensitivity, and long-wavelength emission.