A resorufin-based fluorescent turn-on probe responsive to nitroreductase activity and its application to bacterial detection

Article abstract of DOI:10.1016/j.dyepig.2019.107779

We present two resorufin-based fluorescent turn-on probes responsive to nitroreductase (NTR) activity (NTR probes 1 and 2) and their use toward the detection of bacteria. The probes exhibit a fluorescence turn-on at 586 nm in response to NTR activity, whi

Full text of DOI:10.1016/j.dyepig.2019.107779

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A resorufin-based fluorescent turn-on probe responsive to nitroreductase activity and
its application to bacterial detection
Jung Won Yoon, Sejeong Kim, Yohan Yoon, Min Hee Lee
PII:
S0143-7208(19)31803-0
DOI:
https://doi.org/10.1016/j.dyepig.2019.107779
DYPI 107779
Reference:
To appear in: Dyes and Pigments
Received Date: 29 July 2019
Revised Date: 6 August 2019
Accepted Date: 6 August 2019
Please cite this article as: Yoon JW, Kim S, Yoon Y, Lee MH, A resorufin-based fluorescent turn-on
probe responsive to nitroreductase activity and its application to bacterial detection, Dyes and Pigments
(2019), doi: https://doi.org/10.1016/j.dyepig.2019.107779.
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Graphical Abstract

A resorufin-based fluorescent turn-on probe responsive to
nitroreductase activity and its application to bacterial detection
Jung Won Yoona, Sejeong Kimb, Yohan Yoonb,c,*, Min Hee Leea,*
aDepartment of Chemistry, Sookmyung Women’s University, Seoul 04310, Korea
bRisk Analysis Research Center, Sookmyung Women’s University, Seoul 04310, Korea
cDepartment of Food and Nutrition, Sookmyung Women’s University, Seoul 04310, Korea
*Corresponding author: yyoon@sookmyung.ac.kr (Y. Yoon), minheelee@sookmyung.ac.kr (M.
H. Lee)
Abstract
We present two resorufin-based fluorescent turn-on probes responsive to nitroreductase (NTR)
activity (NTR probes 1 and 2) and their use toward the detection of bacteria. The probes exhibit a
fluorescence turn-on at 586 nm in response to NTR activity, which corresponds to a vivid pink
color. These optical changes are based on the NTR-mediated reduction of the nitro groups in the
probes, which results in the production of highly fluorescent resorufin. The detection ability,
mechanism, and enzyme kinetics of the probes for NTR activity have been thoroughly
investigated. The enzyme efficiencies of the probes were superior to those previously reported.
In addition, NTR probe 1 exhibited an excellent detection ability for NTR with high selectivity
and no background signal when compared to 2. Moreover, NTR probe 1 was applicable for
bacterial detection using a wash-free process because it gave rise to a fluorescence turn-on signal
in response to bacterial NTR activity.

Keywords
Detection of nitroreductase (NTR) activity, fluorescent off-on probe, resorufin dye, bacteria
detection
1. Introduction
Bacteria found in the human body function in the digestive tract, production of vitamins,
destruction of diseased cells, etc [1,2]. However, some bacteria such as Staphylococcus aureus
(S. aureus), Enterococcus faecium (E. coli), Klebsiella pneumoniae (K. Pneumoniae),
Acinetobacter baumannii (A. baumannii), and Pseudomonas aeruginosa (P. aeruginosa), and
their mutations can cause harm to human beings [3–7]. Harmful bacteria also invade the human
body through scars, polluted food, or contact with an infected person. Infiltrated bacteria can
develop complications, leading to death [8]. Therefore, it is important to know exactly where and
how much bacteria are present.
To date, various optical methods have been developed for the detection of bacteria [9–11].
Fluorescence imaging has attracted a great deal of attention due to its high sensitivity and good
spatial resolution. Currently, a number of bacteria targeting fluorescent dyes have been
developed [12–14]. However, most of the dyes have intrinsic fluorescence feature and display
background fluorescent signal, often causing ambiguous analysis of the data in that fluorescent
regions can be caused either by the presence of bacteria or by the presence of the dyes. So, the
dyes should require extensive washing steps for removal of unbound dyes. Therefore, we
envisioned that bacteria specific enzyme-targeted fluorescence turn-on probes will be an
excellent optical tool for bacterial detection.
In this study, we have focused on nitroreductase (NTR), which are flavoenzymes widely
present in bacteria [15]. NTR catalyzes the NAD(P)H-dependent reduction of nitro groups in
nitroaromatic compounds [16,17]. NTR is considered to play an important role in the
detoxification of carcinogens because most nitroaromatic compounds are toxic and carcinogenic
[18]. It has also been reported that NTR activity is increased in human tumor cells and is widely
employed in prodrug activation during chemotherapeutic cancer therapy [19]. However, NTR-

targeted fluorescence turn-on probes that show a detection ability for living bacteria have been
rarely studied.
Herein, we have developed two resorufin-based fluorescent turn-on probes (1 and 2), which
can detect NTR activity. In the presence of NTR activity, the probes exhibit a fluorescence turn-
on signal corresponding to a vivid pink color. In addition, the probes show a highly sensitive
fluorescent off-on response to NTR activity when compared to those previously reported in the
literature [20–27]. Moreover, this system can be used for the detection and imaging of living
bacteria.
2. Material and methods
2.1. Instrumentation
The absorption and fluorescence spectra were recorded on UV-2600 (Shimadzu Corporation,
Kyoto, Japan) and RF-6000 (Shimadzu Corporation, Kyoto, Japan) spectrophotometers,
respectively. Nuclear magnetic resonance (NMR) spectroscopy was recorded on a Bruker
instrument (500 MHz). Low-resolution mass spectroscopic analysis was performed on a Dionex
Ultimate 3000 HPLC system (Thermo Fisher Scientific), and high-resolution mass spectroscopy
(HRMS) was performed on a MS Q-TOF/G6550A instrument (Agilent) at the Korea Basic
Science Institute (Seoul).
2.2. Absorption and fluorescence spectroscopic analyses
The solutions of metal chloride salts (e.g., Na+, K+, Mg2+, and Ca2+), thiols (e.g., glutathione
(GSH), cysteine (Cys), homocysteine (Hcy), NaHS, and dithiothreitol (DTT)), reactive oxygen
species (ROS) (e.g., KO2, H2O2, t-BuOOH, and ClO–), bovine serum albumin (BSA), glucose,
vitamin C, and NADH were prepared using deionized water. A solution of nitroreductase (NTR)
was prepared using phosphate buffer saline (PBS) (10 mM, pH 7.4). The solutions of dicoumarol
and the synthetic probes were prepared in DMSO (HPLC reagent grade). All spectra were
recorded in a PBS solution (10 mM, pH 7.4) containing 1% v/v DMSO at 37 . Excitation was

affected at 564 nm with the excitation and emission slit widths of 5 and 3 nm, respectively.
Fluorescence quantum yields were determined using standard methods with resorufin (Φf = 0.74
in water) as a standard [28].
2.3. Enzyme kinetics of the NTR probes
The enzyme kinetics were performed based on the Michaelis–Menten equation:
ꢁ
ꢂꢃꢄ [ꢅꢆꢇꢅꢈꢉꢊꢈꢋ]
ꢀ =
ꢌꢂ + [ꢅꢆꢇꢅꢈꢉꢊꢈꢋ]
where v is the reaction rate, Vmax is the maximum velocity, [substrate] is the probe’s
concentration, and Km is the Michaelis constant. On this basis, kcat and the turnover number can
be obtained using Vmax/[NTR] and kcat/Km, respectively. These experiments were accomplished
using 1.0 µg/mL nitroreductase, 200 µM NADH, and 1–10 µM of probe 1 or 2 at 37 . The
excitation and emission wavelengths were set at 564 and 586 nm, respectively.
2.4. Synthesis of 1
Resorufin sodium salt (0.24 g, 1.0 mmol) and K2CO3 (0.21 g, 1.5 mmol) were dissolved in dry
DMF (7 mL) and a solution of 4-nitrobenzyl bromide (0.22 g, 1.0 mmol) in dry DMF (3 mL)
was added dropwise to the resulting mixture, which was then stirred at 40 for 3 h. The mixture
was diluted with ethyl acetate (EA) (100 mL) and washed with brine three times. The organic
layer was collected and dried with anhydrous sodium sulfate. After removal of the solvent, the
crude product was recrystallized from EA/diethyl ether to give 1 as an orange solid (0.23 g, 65%).
HR-ESI-MS m/z [M+H]+ calc. 349.07, obs. 349.0832. 1H NMR (500 MHz, DMF-d7 containing a
few drops of DMSO-d6): δ (ppm) 5.55 (s, 2H), 6.29 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 9.8, 2.0 Hz,
1H), 7.27-7.26 (m, 2H), 7.56 (d, J = 9.8 Hz, 1H), 7.95 (dd, J = 8.8, 2.8 Hz, 3H), 8.34 (d, J = 8.7
Hz, 2H). 13C NMR (125 MHz, DMF-d7 containing a few drops of DMSO-d6): δ (ppm) 186.0,
151.5, 150. 5, 149.2, 146.5, 146.0, 144.7, 135.6, 134.6, 132.2, 129.1, 129.1, 127.9, 124.3, 124.2,
114.9, 106.5, 102.1, 69.9.

2.5. Synthesis of 2
Resorufin sodium salt (0.15 g, 1.0 mmol) and K2CO3 (0.13 g, 1.5 mmol) were dissolved in dry
DMF (5 mL) and a solution of 4-nitrobenzyl chloroformate (0.14 g, 1.0 mmol) in dry DMF (2
mL) was added dropwise to the resulting mixture, which was then stirred at 40 for 3 h. The
mixture was diluted with EA (100 mL) and washed with brine three times. The organic layer was
collected and dried with anhydrous sodium sulfate. After removal of the solvent, the crude
product was recrystallized from EA/diethyl ether to give 2 as an orange solid (0.16 g, 63%). HR-
1
ESI-MS m/z [M+H]+ calc. 393.06, obs. 393.0733. H NMR (500 MHz, DMF-d7): δ (ppm) 5.58
(s, 2H), 6.31 (d, J = 2.0 Hz, 1H), 6.88 (dd, J = 9.8, 2.0 Hz, 1H), 7.47 (dd, J = 8.7, 2.5 Hz, 1H),
7.60 (dd, J = 6.2, 3.5 Hz, 2H), 7.86 (d, J = 8.7 Hz, 2H), 7.96 (d, J = 8.7 Hz, 1H), 8.36 (d, J = 8.7
Hz, 2H). 13C NMR (125 MHz, DMF-d7): δ (ppm) 186.0, 153.8, 152.7, 149.9, 149.1, 148.1,
144.6, 143.1, 135.5, 135.1, 131.8, 131.4, 129.3, 127.3, 124.0, 123.5, 119.1, 109.7, 106.6, 69.3.
2.6. Synthesis of 3
Resorufin sodium salt (0.24 g, 1.0 mmol) and K2CO3 (0.21 g, 1.5 mmol) were dissolved in dry
DMF (7 mL) and benzyl bromide (0.12 mL, 1.0 mmol) was added dropwise to the resulting
mixture, which was then stirred at 40 for 4 h. The mixture was diluted with EA (100 mL) and
washed with brine three times. The organic layer was collected and dried with anhydrous sodium
sulfate. After removal of the solvents, the crude product was purified on a silica-gel column
using EA/hexanes (1:1, v/v) as the eluent to give 1 as an orange solid (0.16 g, 54%). HR-ESI-MS
1
m/z [M+H]+ calc. 304.09, obs. 304.0982. H NMR (500 MHz, CDCl3): δ (ppm) 5.18 (s, 2H),
6.32 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 9.8, 2.0 Hz, 1H), 6.88 (d, J = 2.6 Hz, 1H), 7.02 (dd, J =
13
8.9, 2.6 Hz, 1H), 7.46–7.36 (m, 6H), 7.71 (d, J = 8.9 Hz, 1H). C NMR (125 MHz, CDCl3): δ
(ppm) 186.3, 162.7, 149.8, 145.7, 145.6, 135.4, 134.7, 134.3, 131.6, 128.9, 128.6, 128.5, 127.5,
114.3, 106.8, 101.1, 70.9.

2.7. Bacterial cell preparation
E. coli (NCCP15661), S. aureus (KACC10768), A. baumannii (ATCC19606), K. pneumonia
(KACC25306), and P. aeruginosa (NCCP11229) were used in this study. Single colonies of E.
coli (MacConkey Agar; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and S.
aureus (Mannitol Salt Agar; Becton, Dickinson and Company) on selective agar plates were
incubated in 10 mL of tryptone soy broth (TSB; Becton, Dickinson and Company) at 37
overnight. 1 mL aliquots of the cultures were inoculated into 10 mL of TSB and incubated at 37
up to an OD600 of 0.5–0.7. The subcultures were then centrifuged at 1912 ×g for 15 min at 4
. The cell pellets were washed twice with PBS (pH 7.4; 0.2 g of KH2PO4, 1.5 g of
Na2HPO4·7H2O, 8.0 g of NaCl, and 0.2 g of KCl in 1 L of distilled water) and suspended in PBS
to obtain an OD600 of 2.0. A. baumannii, K. pneumonia, and P. aeruginosa were also cultured
and subcultured in 10 mL of Luria-Bertani (LB) broth (Becton, Dickinson and Company) as
described above. Bacterial cell suspensions were then prepared using the same procedure
described for E. coli and S. aureus.
2.8. Fluorescence measurements of bacterial detection using probe 1
1 mL aliquots of the cell suspensions were treated with 10 µM of NTR probe 1 with or without
dicoumarol (0.2 mM), an NTR inhibitor, in microcentrifuge tubes at 37 for 2 h. In addition, to
confirm the florescence change of the NTR probe 1 in dead bacterial cells, 1 mL of the bacterial
cell suspensions in microcentrifuge tubes were heat-treated at 85
for 10 min. The dead
bacterial cells in the microcentrifuge tubes were then treated with NTR probe 1 (10 µM) at 37
for 2 h. The bacterial cells in the microcentrifuge tubes were then lysed upon sonication and the
reactants were centrifuged at 18000×g for 1 min at 25 . 200 µL of the resulting supernatants
were transferred to a SPL 96-well microplate (SPL life science, Kyoungki, South Korea) and the
fluorescence measured using a SpectraMax i3x microplate reader (Molecular Devices, San Jose,
CA, USA) (λex = 564 ±20 nm and λem = 586 ±25 nm).
2.9. Confocal imaging

500 µL aliquots of the E. coli and S. aureus cell suspensions were treated with 10 µM of NTR
probe 1 with or without dicoumarol (0.2 mM) at 37 for 2 h. The bacterial cells in the
microcentrifuge tubes were washed with PBS to remove the unbound dye molecules and treated
with 5 µg/mL of Syto 63 (Invitrogen, Carlsbad, CA, USA) at 37 for 5 min. After
centrifugation at 18000 ×g for 1 min at 25 , the supernatants were removed and the stained
bacterial cells in the microcentrifuge tubes were then transferred onto slide glasses by swabbing.
SlowFade Antifade Reagents (ThermoFisher Sicentific; Waltham, MA, USA) were dropped on
the slide glasses and the samples were then covered with cover glasses. The fluorescence images
of these samples were obtained using a ZEISS LSM 710 Confocal Microscope (63 × oil
immersedoptics) equipped with a high pressure He-Ne lamp and diode laser as the excitation
source (NTR probe 1, λex = 564 ±20 nm and λem = 586 ±25 nm; Syto 63, λex = 657 ±20 nm and
λem = 673 ±25 nm).
3. Results and discussion
3.1. Design principle of the NTR probes
Nitroreductase is involved in the reduction of nitroaromatic compounds to their corresponding
amines with the aid of NADH as an electron source (Scheme 1a) [16,17]. Resorufin dye was
chosen as a framework for our NTR probes because it has an efficient intramolecular charge
transfer (ICT) structure, which exhibits a high fluorescence quantum yield in water (Φf = 0.74)
[28] corresponding to a vivid pink color. In this study, we present two NTR probes (1 and 2)
comprised of resorufin dye conjugated with a p-nitrobenzyl group, where 1 and 2 contain an
ether and carbonate linker, respectively. We also prepared analogue 3, which lacks the nitro
group on the phenyl group (Scheme 1b). The probes were prepared using a simple one-step
reaction in moderate yield (65, 63, and 54%, respectively) (Scheme S1). The structures of 1–3
were confirmed by 1H and 13C NMR spectroscopy, and HR-ESI-MS analysis (Fig. S4–S12).
We envisaged that NTR probes 1 and 2 are non-fluorescent and colorless in an aqueous
condition. However, in the presence of NTR and NADH, the nitro groups of the probes will be
reduced to amines followed by a 1,6-rearrangement elimination, resulting in the production of

highly fluorescent resorufin dye (Schemes 1c, S2, and S3) [29-31]. Thus, NTR probes 1 and 2
can give rise to a fluorescence turn-on signal and vivid pink color in response to NTR activity,
enabling the detection and real-time monitoring of NTR activity. In contrast, analogue 3 lacking
the nitro group on the phenyl group shows no optical change in response to NTR activity (Fig.
S1).
Scheme 1. (a) Enzymatic reduction of nitroaromatic compounds by NTR. (b) The chemical
structures of resorufin, 1, 2, and 3. (c) The proposed detection principle of the probes for NTR
activity.
3.2. Spectroscopic properties of the NTR probes

The absorption and fluorescence spectra of the NTR probes were recorded in the absence and
presence of NTR activity in PBS buffer (10 mM, pH 7.4) containing 1% v/v DMSO at 37 (Fig.
1). A solution of NTR probe 1 displays an absorption at 450 nm with no fluorescent emission,
whereas in the presence of NTR and NADH, probe 1 displays an absorption at 564 nm and
emission at 586 nm (Fig. 1a). A colorless solution of NTR probe 1 turned pink and showed a
strong fluorescence. However, a solution of NTR probe 2 shows absorptions at 450 and 564 nm
and an emission at 586 nm (Fig. 1b). Upon the addition of NTR and NADH to the solution of
NTR probe 2, the absorption at 564 nm and emission at 586 nm were significantly enhanced.
Fig. 1. The absorption and fluorescence spectra of NTR probes (a) 1 and (b) 2 (10 µM,
respectively) in the absence (black line) and presence (red line) of NTR (2.0 µg/mL) and NADH
(200 µM). All spectra were acquired in PBS buffer (10 mM, pH 7.4) containing 1% v/v DMSO
after incubation for 30 min at 37 . Excitation at 564 nm.

The time-dependent fluorescence changes of the NTR probes were monitored in the absence
and presence of NTR activity in PBS buffer (10 mM, pH 7.4) containing 1% v/v DMSO at 37
(Fig. 2). Upon addition of NTR and NADH, a solution of NTR probe 1 promptly displays a
fluorescence increase at 586 nm until 30 min, which was saturated over 90 min. In contrast,
without NTR and NADH, the solution of NTR probe 1 shows no fluorescence signal at 586 nm
even upon incubation for 90 min. A similar fluorescence increase at 586 nm was observed for
NTR probe 2 in the presence of NTR and NADH. However, in the absence of NTR and NADH,
NTR probe 2 itself gave rise to a slight fluorescence increase at 586 nm upon incubation for 90
min. These results indicate that when compared with NTR probe 2, 1 is sensitive to NTR activity
and shows no background fluorescence signal during the detection of NTR activity under
physiological conditions.

Fig. 2. Time-dependent changes in the fluorescence intensity at 586 nm observed for NTR
probes (a) 1 and (b) 2 (10 µM, respectively) upon addition of NTR (2.0 µg/mL) and NADH
(200 µM). All data were acquired in PBS buffer (10 mM, pH 7.4) containing 1% v/v DMSO at
37 . Excitation at 564 nm.
3.3. Sensing mechanism of the NTR probes
To identify the sensing mechanism of the NTR probes presented in Scheme 1, the changes in
the absorption and fluorescence spectra of the probes were compared with those of resorufin, the
expected reaction product (Fig. 3). A solution of resorufin displays absorption and emission at
572 and 586 nm, respectively.30 These are similar to the absorption and emission spectra of NTR
probe 1 observed in the presence of NTR and NADH (Fig. 3a and b). Moreover, a mixed
solution of NTR probe 1 and NTR/NADH was analyzed using ESI-MS spectrometry (Fig. 3c). A
mass peak at 212.08 m/z corresponding to resorufin appeared in the spectrum, while the original
mass peak corresponding to the NTR probe 1 at 349.08 m/z disappeared. Similar results were
also obtained for NTR probe 2 (Fig. S2). These results confirm that the NTR probes undergo a
reduction of the nitro group in response to NTR activity to produce vivid pink and highly
fluorescent resorufin, as proposed in Scheme 1.

Fig. 3. The (a) absorption and (b) fluorescence spectra of resorufin and NTR probe 1 in the
absence and presence of NTR and NADH. All spectra were acquired in PBS buffer (10 mM, pH
7.4) containing 1% v/v DMSO after incubation for 30 min at 37 . Excitation at 564 nm. (c) The
ESI-MS spectrum of the reaction mixture containing NTR probe 1 and NTR/NADH.
3.4. pH effect and selectivity of the NTR probes
The pH effect on the detection of NTR activity using NTR probes 1 and 2 was investigated
(Fig. 4a and b). In response to NTR activity, NTR probe 1 displays a distinctive fluorescence
increase at 586 nm in the pH range of 6–9, which is biologically relevant. In addition, NTR
probe 1 gave no fluorescence signal in the absence of NTR activity. In contrast to NTR probe 1,
NTR probe 2 shows a fluorescence increase at 586 nm in response to NTR activity in the pH
range of 5–11. In addition, NTR probe 2 itself gives rise to a significant fluorescence increase at
pH 7–11, which infers that NTR probe 2 is chemically labile and displays a background

fluorescence signal in the absence of NTR activity. These results indicate NTR probe 1, unlike
NTR probe 2, can provide an efficient fluorescence turn-on response to NTR activity without
interfering the background signal under physiologically relevant pH conditions.
To check the selectivity of the NTR probes toward the detection of NTR activity, their
fluorescence responses were monitored towards various biologically abundant analytes such as
bio-thiols, reactive redox species (ROS), metal ions, bovine serum albumin (BSA), glucose,
NADH, and vitamin C under physiological conditions (Fig. 4c and d). In the presence of NTR
and NADH, NTR probe 1 shows a significant fluorescence increase at 586 nm. In contrast, no
fluorescence increase was observed for the other analytes tested. For NTR probe 2, an increase in
fluorescence was observed not only in response to NTR activity, but also toward ClO– and BSA.
These results revealed that, in contrast to NTR probe 2, NTR probe 1 exhibits a selective
fluorescence off-on response to NTR activity over other biologically abundant analytes.
Therefore, NTR probe 1 can be utilized as an excellent fluorescent probe that can detect and
visualize NTR activity because it gives rise to a highly selective fluorescence turn-on signal
without interference from other biologically relevant analytes and pH.

Fig. 4. The pH effect on the fluorescence increase rate (v) of NTR probes (a) 1 and (b) 2 (10 µM,
respectively) in the absence or presence of NTR (2 µg/mL) and NADH (200 µM). The
fluorescence responses of NTR probes (c) 1 and (d) 2 (10 µM, respectively) toward NADH (200
µM), metal ions (NaCl, MgCl2, KCl, and CaCl2; 1 mM, respectively), thiols (GSH, Cys, Hcy,
NaHS, and DTT; 1 mM, respectively), ROS (KO2, H2O2, t-BuOOH, and ClO–; 100 µM,
respectively), BSA (1 mg/mL), glucose (1 mM), vitamin C (1 mM), and NADH (200 µM) +
NTR (2 µg/mL). All data were acquired in PBS buffer (10 mM, pH 7.4) containing 1% v/v
DMSO with incubation for 30 min at 37 . Excitation at 564 nm.

3.5. Inhibitory test of NTR activity
The detection abilities of the NTR probes toward NTR activity were evaluated in the
presence of dicoumarol, a typical inhibitor of NTR (Fig. 5). Dicoumarol is known to act as a
competitive inhibitor of NADH leading to the inhibition of NTR activity [32,33]. Upon
increasing the concentration of dicoumarol (0, 100, and 800 µM), the absorption and
fluorescence changes observed for the NTR probes were noticeably restricted (Fig. S3). These
results show that the fluorogenic reactions of the NTR probes were mediated by NTR activity.
Fig. 5. The inhibition of the fluorogenic reactions of NTR probes (a) 1 and (b) 2 (10 µM,
respectively) in response to NTR activity (200 µM of NADH and 2 µg/mL of NTR) in the
presence of different concentrations of dicoumarol (0, 100, and 800 µM, respectively). All data
were acquired in PBS buffer (10 mM, pH 7.4) containing 10% v/v of DMSO after incubation for
30 min at 37 . Excitation at 564 nm.
3.6. Enzyme kinetics of NTR to probes 1 and 2
15

The enzyme kinetics of NTR to probes 1 and 2 were investigated by monitoring the
fluorescence increase at 586 nm and the results displayed in Fig. 6. Based on the Michaelis–
Menten equation, the kinetic parameters of NTR to probes 1 and 2 were obtained; maximum
initial reaction rate (Vmax) = 1.5 × 10–4 and 0.011 µMs–1, Michaelis constant (Km) = 3.36 and
14.38 µM, catalyst efficiency (kcat) = 0.87 s–1 and 62.82 s–1, turnover number (kcat/Km) = 0.26 and
4.37 µM–1s–1, respectively. The turnover number represents the number of NTR probes converted
to resorufin per second and thus, it can be considered as the enzymatic efficiency of NTR to the
probes. Moreover, we compared the kinetic parameters (Vmax, Km, kcat, and turnover number) of
NTR probes 1 and 2 with other previously reported probes (Table S1) [20–27]. It was noteworthy
that NTR probes 1 and 2 show a relatively high efficiency for NTR activity when compared to
those previously reported.
Fig. 6. The enzyme kinetics of NTR (0.5 µg/mL) to NTR probes (a) 1 and (b) 2. The Michaelis–
Menten equation was used, where v is the initial reaction rate, [probe] is the substrate’s
concentration, Km is the Michaelis constant, and kcat is the molecular activity number. All data
16

were acquired in the presence of NADH (200 µM) in PBS buffer (10 mM, pH 7.4) containing 1%
v/v DMSO after incubation at 37 . Excitation at 564 nm.
3.7. Detection of bacteria using NTR probe 1
Because probe 1 emits fluorescence, when subjected to NTR, we envisage that NTR probe 1
may be used to detect bacteria that produce NTR during their growth. To examine the use of
NTR probe 1 as a dye for detecting various pathogenic bacteria, the fluorescence intensities of
the bacterial cells were measured after they were treated with NTR probe 1 with or without an
NTR inhibitor (dicoumarol). As a result, treating the bacterial cells with NTR probe 1 induced an
increase in the fluorescence intensity, and the addition of dicoumarol reduced the fluorescence
intensity (Fig. 7a). Furthermore, the fluorescence intensity was reduced in dead bacterial cells,
which do not produce NTR (Fig. 7b).
Fig. 7. The detection of live bacteria (a) and dead bacteria heated at 85 for 10 min (b) using
NTR probe 1. The fluorescence was measured after the bacterial cells were stained using 10 µM
NTR probe 1 without (black bar) or with (light grey bar) dicoumarol (0.2 mM). A comparison of
the fluorescence intensities observed between live (dark grey bar) and dead (white bar) bacteria
cells. PA: P. aeruginosa, KP: K. pneumonia, EC: E. coli, SA: S. aureus, and AB: A. baumannii.
(λex = 564 ±20 nm and λem = 586 ±25 nm).
To visualize the detection of live bacterial cells using NTR probe 1, confocal images with red
color were obtained. To prove if there were bacterial cells, Syto 63, which stains live bacterial
17

cells with blue color, was used. The confocal images and fluorescence intensities show that NTR
probe 1 detected live E. coli and S. aureus cells, but this depended on the treatment of
dicoumarol (NTR inhibitor) (Fig. 8). These indicate that NTR probe 1 can afford a strong
fluorescence response to bacterial NTR activity and thus, it is applicable for the detection of live
bacterial cells.
Fig. 8. Confocal images of E. coli (a) and S. aureus (b) obtained using a ZEISS LSM 710
confocal microscope (63 × oil immersed optics) (NTR probe 1, λex = 564 ±20 nm and λem = 586
±25 nm; Syto 63, λex = 657 ±20 nm and λem = 673 ±25 nm). The bacterial cells were treated with
18

10 µM NTR probe 1 without or with an NTR inhibitor (0.2 mM dicoumarol) at 37 for 1 h.
Syto 63 was also used to stain all live cells.
4. Conclusions
We have developed two resorufin-based fluorescent turn-on probes (NTR probes 1 and 2)
that can detect bacterial nitroreductase (NTR) activity. In response to NTR activity, the probes
give rise to a significant fluorescence increase at 586 nm corresponding to a vivid pink color.
This is based on a NTR-mediated fluorogenic reaction leading to the production of highly
fluorescent resorufin. NTR probe 1 exhibited a superior detection ability toward NTR activity
when compared to NTR probe 2. In addition, it has been demonstrated that NTR probe 1 is
applicable for the detection of various pathogenic bacteria. We thus proposed that NTR probe 1
will be used as a fluorescent turn-on probe for bacterial detection with high selectivity and
sensitivity, wash-free imaging and moderate detection time.
Acknowledgments
This research was supported by National Research Foundation of Korea (NRF)
(2018R1C1B6006110, M.H.L.).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:
http://doi.org/ddd
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Highlights
•
Resorufin-based fluorescent turn-on probes 1 and 2 were developed for detecting
nitroreductase (NTR) activity.
•
•
The probes showed a fluorescence turn-on signal with a vivid pink color to NTR activity.
The probes exhibited moderate response time to NTR activity with a high enzyme
efficiency.
•
•
The NTR probe 1 exhibited a superior detection ability for NTR activity to probe 2.
The NTR probe 1 can act as a fluorescent turn-on probe for bacterial detection.