Naphthalimide-Based DNA-Coupled Hybrid Assembly for Sensing Dipicolinic Acid: A Biomarker for Bacillus anthracis Spores

Article abstract of DOI:10.1021/acs.langmuir.8b00340

We have designed and synthesized a novel, water-soluble naphthalimide-histidine receptor (1) with excellent fluorescent properties. Functioning of the synthesized receptor was performed through developing their DNA-receptor hybrid assembly (DRHA), which has shown significant changes in the emission profile upon interactions with dipicolinic acid (DPA), a biomarker for Bacillus anthracis spores. DRHA showed fluorescence enhancement upon binding with DPA with the characteristic of internal charge transfer. It is notable that this assembly exhibited a significant limit of detection (12 nM) toward DPA. The mechanism of sensing was fully defined using ethidium bromide (EtBr) interaction studies as well as Fourier transform infrared spectroscopic analysis, which describes the binding mode of DRHA with DPA. This assembly selectively interacts with DPA over other anions, common cellular cations, and aromatic acids in aqueous media.

Full text of DOI:10.1021/acs.langmuir.8b00340

Article
Cite This: Langmuir XXXX, XXX, XXX−XXX
pubs.acs.org/Langmuir
Naphthalimide-Based DNA-Coupled Hybrid Assembly for Sensing
Dipicolinic Acid: A Biomarker for Bacillus anthracis Spores
†
‡
,†
Meenakshi Verma, Navneet Kaur, and Narinder Singh*
†Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar, Punjab, India
‡Department of Chemistry, Panjab University, Chandigarh 160014, India
*
S Supporting Information
ABSTRACT: We have designed and synthesized a novel,
water-soluble naphthalimide-histidine receptor (1) with
excellent fluorescent properties. Functioning of the synthe-
sized receptor was performed through developing their DNA−
receptor hybrid assembly (DRHA), which has shown
significant changes in the emission profile upon interactions
with dipicolinic acid (DPA), a biomarker for Bacillus anthracis
spores. DRHA showed fluorescence enhancement upon
binding with DPA with the characteristic of internal charge
transfer. It is notable that this assembly exhibited a significant
limit of detection (12 nM) toward DPA. The mechanism of
sensing was fully defined using ethidium bromide (EtBr)
interaction studies as well as Fourier transform infrared spectroscopic analysis, which describes the binding mode of DRHA with
DPA. This assembly selectively interacts with DPA over other anions, common cellular cations, and aromatic acids in aqueous
media.
14−16
INTRODUCTION
same.
DPA detection, thus, may provide good progression
■
for counting bacteria spore content in the affected material.
Besides the toxicity of bacterial spores, even DPA alone, if
inhaled, can lead various systemic or central nervous system
effects such as memory difficulties, abnormal fatigue, or
Bacterial contamination in various resources of foodstuff or
water necessitates continued development to discover rapid and
1
specific techniques for detecting the pathogens involved. The
growth of highly sensitive and selective probes for the
recognition and measurement of these pathogens, in vitro, is
one of the most necessary tools in biological, chemical, and
17
dizziness. The present literature reports revealed various
biological and chemical techniques to detect DPA through
18−20
21,22
2
polymerase chain reaction
and immunoassays;
how-
environmental science. The current scenario leads toward
ever, these are affected with some drawbacks such as prolonged
cycles, difficult process, costly reagents, and specialized
significant progress in the field of biosensors that results in the
development of more specific, robust, and economic methods
by incorporating emerging variety of techniques from diverse
23−26
analysis.
A list of important chemical methods used for
1
identifying B. anthracis includes vibrational spectroscopy
disciplines.
[
Fourier transform infrared (FTIR), Raman, and surface-
Dipicolinic acid (DPA) is a unique constituent of bacterial
spores of Bacillus anthracis, accounting for 5−15% of the dry
mass of the spores and furthermore is unique to B. anthracis.
Consequently, DPA plays an important role as a potential
2
7−31
32
enhanced Raman],
potentiometric sensors, high performance-liquid chromatog-
raphy, and pyrolysis mass spectrometry, and optical
detection. The optical methods/luminescence techniques
have currently attracted the interest of the researchers, as
these are rapid, highly sensitive and selective, and economically
capillary zone electrophoresis,
33
3
4
35
3
−5
biomarker for B. anthracis.
B. anthracis is a Gram-positive,
rod-shaped, aerobic and facultative anaerobic, and spore-
forming bacterium and is one of the most hazardous biological
warfare agents because of the possibility of high disaster and
mass destruction. This has already been used as a warhead by
military and terrorist groups.
36−41
viable and offer easy operation.
6
In general, optical sensing of bacterial spores is based on the
7
−10
detection of DPA and is a major component of Bacillus
However, its detection is not
4
2
spores. It is evident from literature reports that the
lanthanides showed good potential for DPA detection because
of their distinctive and quick complexation with DPA.
an easy task, as it takes upto 60 days for anthrax symptoms to
appear in humans.
11−13
Therefore, developing a detection
method for the biomarker of this infectious agent has been the
key point of current research that can work in an environment
as well as in the aqueous system. After the anthrax attacks in the
United States, the researchers are emphasizing on developing a
precise, rapid, real-time, and field-ready detecting system for the
Received: January 31, 2018
Revised: March 26, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.langmuir.8b00340
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Article
Scheme 1. Synthesis Scheme of Receptor 1
However, as per reports, the fluorescence intensity of
lanthanides is recognized to be responsive to external
conditions, for example, the preparation method, apparatus
setup parameters, ionic concentration, and the nature of target
of DRHA toward DPA has been investigated in tap water, canal
water, and rainwater.
EXPERIMENTAL SECTION
2
6,43
■
moiety.
Consequently, it becomes the need of the hour to
General Information. 1,8-Naphthalic anhydride, bromine, L-
histidine, and ethylenediamine were purchased from Sigma-Aldrich/
local suppliers and were used without further purification. Milli-Q
water (18.2 MΩ·cm at 25 °C) was used to prepare all aqueous
solutions. Purified solvents of commercial grade were purchased from
find some alternatives to overcome these drawbacks. Herein,
we report a naphthalimide-based DNA receptor hybrid
assembly (DRHA) for detection of DPA. Naphthalamide is
the fluorophore tag of choice because of its outstanding
photophysical properties that include high extinction coef-
ficient, photostability, and relatively long emission wave-
1
13
Loba, Spectrochemicals, and Aldrich. The H NMR and C NMR
spectra were recorded on the Jeol Instrument working at 400 MHz for
1
13
4
4
H NMR and at 100 MHz for C NMR. All chemical shifts were
length. The strong emission character of 1,8-naphthalimide
derivatives in organic and aqueous media is thought to be due
recorded in ppm relative to tetramethylsilane as an internal reference.
The UV−vis absorption spectra were recorded on a Shimadzu 2400
spectrophotometer using cuvettes of 1 cm path length. The
fluorescence experiments were conducted at room temperature on a
PerkinElmer LS-55 Spectrophotometer with a fixed scanning speed
and emission slit width (10 nm). Particle size was determined by
dynamic light scattering (DLS) using the external probe feature of the
Metrohm Microtrac Ultra Nanotrac particle size analyzer. Time-
resolved fluorescence decay studies were performed on a PicoQuant
FluoTime 300 high-performance fluorescence lifetime spectrometer.
FTIR spectra images were obtained using a Bruker photospectrometer.
Circular dichorism (CD) spectra were recorded on a JASCO, J-1500
circular dichroism spectrophotometer between 400 and 200 nm in the
continuous scanning mode (200 nm/min).
45
to their internal charge-transfer (ICT) excited-state transition.
,8-Naphthalimide derivatives having an aromatic core
1
moiety and bearing a basic side chain offer good DNA binding
affinity either by intercalation or groove binding. The synthetic
strategy allows modifications with a variety of functional groups
on both sides: the aromatic “naphthalene” moiety and the “N-
4
6,47
imide site”.
The carbonyl group of naphthalimide has an
electron-withdrawing nature and hence facilitates the intro-
duction of electron-donating groups (e.g., amines) in the
48
molecular design. The presence of an electron-donating
amine group in the 3- or 4-position provides a green-emitting
fluorophore with tremendous photochemical stability and good
Synthesis and Characterization of 3. The bromination of 1,8-
naphthalic anhydride (6) was achieved as per the method described in
4
5
quantum yields. A system containing naphthalimide as its
core molecule has been used tremendously not only as
5
1,52
the literature.
Briefly, to a solution of 1,8-naphthalic anhydride
45
(1.98 g, 10.0 mmol) in KOH (2.8 g in 12 mL water), 2 mL of bromine
was added drop-wise at room temperature. The reaction mixture was
stirred at room temperature for 30 min and then heated to 60 °C for 6
h. On the completion of the reaction, light brown colored precipitates
separated out. The reaction mixture was acidified with concentrated
hydrochloric acid (HCl), and the solid residue was then filtered. The
crude product was further heated with 5% NaOH solution and filtered,
and the filtrate was neutralized with HCl solution. Solid precipitates
separated out which were filtered and washed with cold water and
dried to get yellow-brown powder of 4-bromo-1,8-naphthalic
anhydride (5). To synthesize 3, compound 5 (2.77 g, 10 mmol)
was suspended in acetic acid, and then L-histidine (1.55 g, 10 mmol)
was added to it. The resulting solution was then heated to 80 °C for 8
h and cooled to room temperature after completion of the reaction. Ice
cooled water was added to this solution and filtered to get dark brown
solid of the compound 3 which was washed with water a number of
colorimetric and luminescent sensors for ions and bio-
11
molecules but also in cellular imaging applications and
49
generation of fascinating supramolecular assemblies. These
derivatives have also been used in numerous other applications,
for example, as luminescent probes for selective staining of live
cells, stimuli-responsive hydrogels, and in photochemical
4
5
welding of tissues. Interestingly, their properties are
extendable as tunable dye lasers, polymerizable materials, and
50
electroluminescent organic diodes in thin films.
In the present work, the structure design utilized the
structural benefits of naphthalimides, and the synthesized
assembly is also appended with the following: (a) histidine
moiety that possibly offers solubility in aqueous media and (b)
the aliphatic amine group owing to its good binding capabilities.
In comparison to the previously known DPA sensors, the
naphthalimide-based optical sensor seems more lucrative owing
times to get rid of any trace amounts of acetic acid. Yield = 82%. mp =
1
1
83−185 °C. H NMR (400 MHz, DMSO-d ): δ 8.55 (t, J = 8 Hz,
6
26
2H, ArH), 8.29 (d, J = 8 Hz, 1H, ArH), 8.20 (d, J = 8 Hz, 1H, ArH),
.99 (t, J = 8 Hz, 1H, ArH), 7.34 (s, 1H, His-ArH), 6.65 (s, 1H, His-
ArH), 5.73 (m, 1H, His-CH), 3.41 (m, 2H, His-CH2). C NMR (100
to its fluorescence properties and ease of use. To the best of
our knowledge, this is the first report in which the
naphthtalimide-based probe is used as the optical detector
against DPA. The developed DRHA has shown an active
response in an aqueous medium; therefore, the practical utility
7
13
MHz, DMSO-d ): δ 171.13, 163.01, 135.22, 133.53, 132.59, 132.07,
6
131.96, 130.37, 130.09, 129.52, 128.75, 122.79, 117.37, 54.09, 26.30.
Anal. Calcd for C H BrN O : C, 52.19; H, 2.95; Br, 19.24; N, 10.19;
18
12
3
4
B
DOI: 10.1021/acs.langmuir.8b00340
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Figure 1. (A) Schematic diagram showing the interaction of organic receptor 1; (B) changes in fluorescence intensity of receptor 1 (1 μM) upon the
successive addition of DNA (0−10 μM) in aqueous medium (λ = 460 nm); (C) typical time-resolved fluorescence decay profile of receptor 1 and
ex
the same receptor in the presence of 10 μM salmon sperm DNA; and (D) circular dichroism (CD) spectra of salmon sperm DNA incubated 18 h
with receptor 1 at different [receptor]/[DNA] ratios at 37 °C. (E) Effect of the increasing concentration of receptor 1 on the relative viscosity of
salmon sperm DNA at 25 °C. (F) Variation in melting temperature of DNA on the addition of receptor 1.
O, 15.40. Found: C, 52.23; H, 2.95; Br, 19.24; N, 10.19; O, 15.40. MS
μM) to the fixed amounts of the receptor (1 mL). The fluorescence
measurements were performed, maintaining the excitation and
emission band slit width of 10 nm. A fluorescence-free quartz cell of
1 cm path length was used. CD spectra of DNA alone and in the
presence of receptor 1 were recorded at room temperature in the
wavelength range of 200−700 nm, at different compound/DNA ratios,
however, keeping the DNA concentration constant. The optical
chamber of the CD spectrometer was deoxygenated with dry nitrogen,
and samples were held under nitrogen atmosphere during the
measurements. Viscosity measurements were carried out using
Ostwald’s viscometer suspended vertically in a thermostat at 25 °C.
The flow time was measured with a digital stopwatch, and the sample
was tested three times to get an average calculated time. Data obtained
+
(EI): m/z 414.07 (M + 1).
Synthesis and Characterization of 1. Compound 3 (0.413 g, 1
mmol) was refluxed overnight with ethylenediamine (2) (10 mmol)
using ethanol as the reaction solvent. Reaction was monitored with
thin-layer chromatography (TLC) and on completion of reaction, the
solvent was evaporated under vacuum. The crude product was then
purified using column chromatography and collected as a yellow solid.
Yield = 85%. mp = 169−171 °C; FT-IR ν: 2981, 2939, 2831, 2252,
−1 1
1
705, 1640, 1527, 1350, 1172, 1107, 1014, 948 cm . H NMR (400
MHz, DMSO-d ): δ 8.47 (d, J = 12 Hz, 2H, ArH), 8.24 (m, 1H, ArH),
6
8
1
.15 (d, J = 8 Hz, 1H, ArH), 7.93 (t, J = 8 Hz, 1H, ArH), 7.30 (d, J = 8,
H, His-ArH), 6.47 (d, J = 12 Hz, 1H, His-ArH), 5.45 (m, 1H, His-
0
1/3
CH), 3.49 (m, 2H, His-CH ), 3.37 (m, 2H, −CH ), 3.25 (t, d = 12 Hz,
were plotted as (η/η ) versus [compound]/[DNA] ratio, where η
2
2
13
0
2
1
7
4
H, −CH ). C NMR (100 MHz, DMSO-d ): δ 163.12, 134.91,
34.73, 132.93, 132.33, 132.10, 131.91, 131.52, 130.26, 129.36, 128.73,
9.72, 56.58, 26.88, 23.31. Anal. Calcd for C H N O : C, 61.06; H,
and η denote the specific viscosity of salmon sperm DNA in the
2
6
presence and absence of the receptor 1, respectively, and their values
were calculated using formula (t − t )/t , where t denotes the observed
20
19
5
4
b
b
.87; N, 17.80; O, 16.27. Found: C, 61.11; H, 4.81; N, 17.85; O, 16.23.
flow time in the presence of receptor 1 and t stands for the flow time
b
53,54
+
MS (EI): m/z 394.21 (M + 1).
of buffer alone.
DNA melting experiments were carried out by
Intercalation of the Receptor with DNA. The solution of
receptor 1 was prepared at 1 mM concentration in water and sonicated
well before recording the spectra. Receptor 1 was freely soluble in
water, and hence, all binding studies were performed at pH 7.5 using
HEPES buffer. A stock solution of salmon sperm DNA was prepared
by dissolving DNA in phosphate buffer. Absorption spectroscopic
changes were observed by adding DNA in incremental amounts (0−10
μM) to the fixed amounts of the receptor (1 μM). The fluorescence
changes were observed with the incremental addition of DNA (0−10
observing the absorbance of salmom sperm DNA at 280 nm at various
temperatures in the absence and presence of testing receptor 1 with a
ramp rate of 1 °C/min in a phosphate buffer (pH 7.0) on a Shimadzu
spectrophotometer equipped with a Peltier thermoregulator. The
transition midpoint of the melting curve was assigned as T . The
m
particle size of aggregates of receptor 1 alone as well as in the presence
of DNA was determined with DLS using the external probe feature of
the Metrohm Microtrac Ultra Nanotrac particle size analyzer. The data
revealed that the particle size lies in the μm range.
C
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Figure 2. Recognition of DPA with DNA−receptor hybrid assembly (DRHA): (A) changes in absorption spectra of DRHA upon the successive
addition of DPA (0−10 μM) in aqueous medium; (B) changes in the fluorescence intensity of DRHA upon the successive addition of DPA (0−10
μM) in aqueous medium (λ = 430 nm), and emission from solution under UV light is shown in the inset of the figure (inset); (C) linear regression
ex
graph between concentrations of DNA added to receptor 1 (decrease in fluorescent intensity) and concentrations of DPA added to DRHA (increase
in fluorescent intensity); (D) competitive binding studies of DRHA containing DPA over other selected anions; (E) plot of the intensity ratio of
DRHA and DPA at different concentrations as a function of time in seconds; (F) effect of pH on DRHA (1 μM) alone and in the presence of DPA
in an aqueous system.
DPA Recognition Using DRHA. The DRHA was used to
recognize DPA (dipicolonic acid). The solution of organic receptor 1
was prepared at 1 mM concentration in the buffer and sonicated well
before recording the spectra. All binding studies were performed at pH
7.5 using HEPES buffer. The stock solutions of DNA and DPA were
prepared at a concentration of 1 mM (pH 7.5, HEPES buffer). The
real sample analysis experiment was based on evaluating the
functioning of the sensor in various environmental samples. For this,
water samples from various sources were collected. To calculate the
detection limit, a graph was plotted between the concentration and the
fluorescence intensity. The detection limit was calculated by using the
formula: DL = (3 × SD)/m.
Figure 3. Selectivity test of DRHA for DPA: detection over several
amino acids, aromatic ligands, and cellular cations. The concentrations
of all the compounds are the same, and measurements are performed
under identical conditions.
RESULTS AND DISCUSSION
■
51,52
Synthesis and Characterization of the Receptor.
Receptor 1 has been designed to target the recognition
properties for the desired analyte, and the receptor design is
fabricated with the histidine group that may impart the water
solubility to the receptor and the naphthalimide unit as
fluorescent signaling subunits. The naphthalimide unit is
articulated with the amine group for binding with the
biomarker, and it is envisioned that once the biomarker binds
reported in the literature.
Compound 3 was synthesized
by reacting 5 with 1 equivalent of L-histidine in acetic acid.
After 8 h of heating at 80 °C, the reaction mixture was cooled
to room temperature, which yielded the precipitates. The
precipitates were filtered and washed with an excess of water to
remove any traces of acetic acid. In next step, the target
receptor 1 was synthesized by reacting compound 3 with an
excess of ethylenediamine in ethanol under reflux. The progress
of the reaction was monitored with TLC, and upon completion
of reaction, the crude product was purified using column
chromatography. Spectroscopic data confirmed the formation
of receptor 1 (Figures S1−7).
with the −NH group, this binding event will modulate the
2
fluorescence signature of the naphthalimide moiety. The
synthesis of the desired receptor was achieved through the
bromination of 1,8-naphthalic anhydride using a method
D
DOI: 10.1021/acs.langmuir.8b00340
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Table 1. Analysis of Spiked Samples of DPA in Real Water
Samples (n = 3) Using Synthesized DRHA
Table 2. Intraday and Interday Precision and Accuracy of
Analytes (n = 4)
ab
DPA added (nM)
0
DPA found (nM)
% recovery
nominal
concentration
measured average
tap water
canal water
rain water
N.D.
0
(
nM)
concentration (nM)
SD
RSD % relative error %
2
4
0
2
4
0
2
4
0
0
18.13 ± 0.05
38.91 ± 0.05
N.D.
95.65
97.27
0
Intraday Assay
2
4
6
8
0
0
0
0
18.24
36.72
56.25
74.45
0.76
1.39
1.57
1.96
4.21
3.80
2.79
2.63
1.18
1.31
1.78
1.89
0
0
18.85 ± 0.05
37.57 ± 0.05
N.D.
94.25
93.92
0
Inter-Day Assay
0
0
18.41 ± 0.05
38.46 ± 0.05
92.05
96.15
2
4
6
8
0
0
0
0
17.24
36.32
54.65
73.95
0.78
1.04
1.70
1.43
4.53
2.87
3.11
1.94
1.10
1.74
1.60
2.57
a
Recovery = (amount found in the spike sample − amount found in
the original sample) × 100/amount added. N.D.: not detected.
b
Intercalation of the Receptor with DNA. The synthe-
sized organic receptor (1) was tested for its binding affinity
with DPA (10 μM) through the modulation of absorption and
fluorescence signatures of 1 with respect to the varied
concentrations of DPA. However, contrary to our expectation,
insignificant changes were detected in absorption and emission
spectra of the receptor 1 upon the addition of DPA. The most
probable reason expected is the hindrance of amide bond
formation between the receptor 1 and DPA. However, the
recent account of Silverman revealed that such bond formation
can be modulated in the presence of DNA. Thus, in the
present work, we decided first to understand the intercalation
of receptor 1 with DNA, and eventually, the DRHA will be
used to sense the biomarker. To explore the binding mode of
DNA with receptor 1, various studies have been performed, and
these studies proposed the intercalating nature of receptor 1
with DNA. Figure 1A presents the schematic diagram showing
the interaction of the organic receptor with DNA. To support
this intercalation, spectroscopic analysis was performed, where
the UV−vis absorption spectroscopy has provided important
information on the mode of binding of molecules to DNA. The
addition of salmon sperm DNA (5 equiv) to the solution of 1
(1 mL) showed modulation in the absorbance and emission
profile of the receptor 1. To examine the binding approach
between DNA and receptor 1, titrations were carried out in
which the concentration of the receptor was kept constant (1
mL) and the DNA concentration was varied from 0 to 10 μM
and the titrations were monitored with UV−vis absorption
spectroscopy. Upon the incremental addition of DNA, the
hypochromic shift has been observed without any band shift in
the absorption spectra of 1 (Figure S8). The observed
“hypochromic” spectral changes suggested that receptor 1
exhibited good binding tendency for salmon sperm DNA. It is a
well-known fact in the literature that hypochromism is a
55
56,57
characteristic property of intercalating molecules.
Further,
the extent of the hypochromism is related to the strength of
intercalative interaction, where this mode of binding comprises
the stacking interaction between an aromatic chromophore and
the base pair of DNA. Fluorescence spectroscopy is one of the
most precise methods for studying the comparative binding of
Figure 4. (A) Diagrammatic representation of the mechanism of sensing of DPA with DRHA; (B) ethidium−bromide interaction experiment
showing interactions between DRHA and DPA; and (C) IR spectra of DRHA alone and in the presence of DPA.
E
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Figure 5. (A) Absorption and (B) emission spectra of DRHA showing its short-term, freeze−thaw, and long-term stability.
a
Table 3. Comparison of the Limit of Detection of Fluorescent Sensors for the Detection of DPA as a Biomarker of B. anthracis
fluorescent sensor
linear
LODs
detection in water
yes
real sample analysis
interference
ref
Eu−GNPs
Tb−GNPs
1 μM
72
Eu−PVA film
Tb−silica NPs
Tb/Eu@bio-MOF
Tb−polymer
FA:Tb−EDTA
FA:Eu−EDTA
AgNPs−Tb
Tb/Eu−MOFs
Tb−CDs
non-linear
100 nM
56.6 nM
34 nM
10 nM
8.2 nM
20.9 nM
6.7 nM
4.55 nM
5 nM
yes
yes
yes
no interference
no interference
no interference
no interference
no interference
66
64
73
68
69
yes
yes
0−20 μM
40 nM to 10 μM
50−700 nM
0.005−1.2 μM
0.25−20 μM
0−9.5 μM
yes
yes
yes
yes
yes
yes
yes
no interference
no interference
no interference
no interference
no interference
70
26
71
65
CDs
79 nM
12 nM
yes
yes
DRHA
this work
a
MOFs: metal−organic frameworks. GNPs: gold nanoparticles.
small molecules to DNA as fluorescence quenching measure-
intensity of both bands owing to disturbance of the stacking of
the nucleobases on intercalation. Upon the addition of
18,51
53
ments can be used to evaluate binding.
The interaction
between receptor 1 and salmon sperm DNA in an intercalation
mode causes a strong fluorescence quenching. Receptor 1 with
λmax = 545 nm (λex = 460 nm) showed quenching with the
successive addition of DNA (0−10 μM) (Figure 1B). The time-
resolved fluorescence decay behavior has been monitored
under differential circumstances to achieve a deeper insight into
the photophysical properties of receptor 1 and its modulation
in the presence of DNA. The fluorescence lifetime indicates the
average time of the excited state of any molecule in its excited
receptor 1 to salmon sperm DNA, the CD spectrum shows
increase in intensities of both the positive and negative bands,
proposing that this receptor binds with DNA through
intercalation (Figure 1D). The viscosity experiment is a
useful tool to detect binding modes between DNA and small
molecules, which is generally performed to clarify the mode of
binding. Ethidium bromide, being one of the well-known DNA
intercalators, causes the DNA helix to increase its length by
61
separating its base pairs to adjust itself, resulting in net viscosity
5
8
55,56
state. Figure 1C represents the time-resolved fluorescence
decay profile of receptor 1 under varied experimental
conditions. The results revealed a single exponential decay for
receptor 1 emissions, and the lifetime was found to be 4.7 ns.
However, the receptor intercalated with DNA revealed that the
decay was at 4.26 and 2.08 ns and hence offers a biexponential
response. It confirms energy transfers in the excited state and
increase.
Though a compound that attaches electrostatically
via the sugar-phosphate backbone generates bends in the DNA
helix which reduce its effective length and hence change its
viscosity, DNA groove binding under the same experimental
environment usually results in little or no effect on DNA
56,57
viscosity.
To know the type of binding between synthesized
receptor and DNA, viscosity measurements has also been
executed using the Ostwald viscometer. The experiments were
conducted by adding appropriate amounts (0−120 μM) of
receptor 1 into the viscometer to give a particular ratio (r =
[receptor]/[ DNA]), while keeping the salmon sperm DNA
concentration constant. The flow time was determined using a
digital stopwatch, and the mean values of three independent
measurements were used to evaluate the average relative
viscosity of the sample.
59
could be static or dynamic. CD is another technique used to
expose the changes in the DNA conformation during
6
0
molecule−DNA interactions. To confirm whether the
synthesized receptor is capable of inducing DNA conforma-
tional changes, CD measurements were recorded in the 200−
500 nm range. The DNA concentration was kept constant
during the experiment, while that of the receptor kept on
changing. The CD spectrum of salmon sperm DNA has two
bands, the negative band at ∼245 nm (helicity) and a positive
band at ∼275 nm (base stacking). These bands in the CD
spectrum emanate from the base stacking interactions and the
double helical supra structure of the polynucleotide, thereby
0
1/3
Plots of obtained data are represented as (η/η ) versus
0
[receptor]/[DNA] ratio, where η and η are the specific
viscosity of salmon sperm DNA in the presence and absence of
the receptor 1, respectively. Relative viscosities of salmon
53
0
54
providing asymmetric environments for the nucleobases.
sperm DNA were calculated from (η/η ). The data shown in
Figure 1E exhibited a notable change in relative specific
viscosity, indicating intercalative binding of receptor 1 toward
salmon sperm DNA. Further, the high temperature can destroy
Simple groove binding or electrostatic interactions of DNA and
receptor result in slight perturbation of the original CD profile
of DNA. However, an intercalating molecule enhances the
F
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the double helix structure of DNA and transform it into a single
μM of DPA solution was added to each flask. Tetrabutylam-
3
−
−
−
−
−
helix at the melting temperature (T ). Interaction of small
monium anion salts (PO , CH COO , ClO , HSO , NO ,
m
4 3 4 4 3
−
−
−
−
molecules with DNA can affect the melting temperature by
Cl , Br , I , F ) were used as potential interference entities as
the DPA is dicarboxylate and with the modulation of pH, it may
offer the anionic species. Each solution was kept for
equilibrium, these solutions were shaken after a regular time
interval, and finally, the fluorescence measurements were
performed with each solution independently. Comparison of
fluorescence spectra of DRHA + DPA alone and of DRHA +
DPA in the presence of various anions showed that there is no
interference from the other anions and the sensor is highly
selective for DPA (Figure 2D).
62
increasing it about 5−8 °C, which can be justified on the basis
of intercalation binding that provides extra stability to the DNA
helix. Nonintercalative bindings do not show such an effect.
DNA melting studies further support the DNA intercalating
nature of receptor 1. The interactions of synthesized receptor 1
with salmon sperm DNA were determined through thermal
denaturation experiments. The value of T was changed to a
m
notable extent. For pure salmon sperm DNA, the melting
temperature is 87.5 °C which increased to 94.3 °C upon the
addition of receptor 1. This supports the fact that the
interaction between DNA and receptor 1 is intercalative
Although the sensor has offered high selectivity, sensitivity is
another important criterion for the successful operation of the
sensor: short response time is mandatory for a sensor meant for
real-time applications. To study the response time of DRHA for
sensing of DPA, the fluorescence spectra were recorded with
solutions containing different concentrations of DPA (3.0, 6.0,
and 9.0 μM) to the solutions of DRHA (1.0 μM), and each
solution was analyzed as a function of time. The response time
of DRHA to detect DPA is concentration-independent, as the
time required to reach equilibrium does not affect DPA
concentrations. The interpretation of results revealed that after
60 s, the fluorescence intensity of all three solutions is
independent of time; in other words, the response time of
DRHA for DPA is less than 60 s (Figure 2E). To gain more
insights about the application of the sensor, the performance of
the fluorescent sensor for target moiety was evaluated under
varied pH conditions. This was evaluated by monitoring the
effect of pH on the fluorescence response of DRHA and DRHA
+ DPA. During these studies, both acidic and basic titrations
were conducted by changing pH of DRHA and DRHA + DPA
solutions independently using sodium hydroxide/hydrochloric
acid. The pH window ranging from 6.0−9.0 for DRHA and
DRHA + DPA both were found to be best for the operation of
the sensor (Figure 2F).
(
Figure 1F). The particle size of aggregates of receptor 1 has
been determined from DLS histograms, and it has been found
that the size of aggregates of 1 is 0.57 μM. The effect of the
addition of a fixed amount of DNA on the size of these
aggregates has also been determined. The size increased from
0
.57 to 0.66 μm. It can be concluded that the size increase on
the addition of DNA to the host receptor is due to aggregation
(
Figure S9).
DPA Recognition Using DRHA. To evaluate the
recognition profile of the DRHA, it was exposed to different
concentrations of DPA in an aqueous solution and absorption,
and the emission profile of the solutions has been tested against
the increasing amount of DPA (0−10 μM). The absorption
spectra of DRHA showed an absorbance band at 430 nm,
which shifted to 451 nm on the addition of 10 μM of DPA. The
titration between DRHA and DPA showed a distinctive trend
when the titration was monitored with UV−vis absorption
spectroscopy. With the increasing concentration of DPA, the
absorbance band showed hypochromic as well as bathochromic
shift up to 445 nm. Beyond this, the band showed an upward
shift along with bathochromic (Figure 2A). This response is
well-explored for the intramolecular charge transfer (CT)
behavior of naphthalimide-based moieties which are widely
used in fields such as sensing, intracellular biomarkers, or
To investigate the selective utility of DRHA, we further
performed competitive binding assay: a number of possible
interference moieties have been selected including glycine, L-
aspartic acid, methionine, cysteine, trypsin, tyrosine, and
phenylalanine as representative amino acids as well as a
collection of different competitive protic/bidentate substrates/
aromatic acids (e.g., catechol, adenosine triphosphate, choles-
terin, glucose, benzoic acid, 2-picolinic acid, 3-aminobenzoic
acid, 2,6-dihydroxy benzoic acid, isophthalic acid, p-toluic acid,
and nicotinic acid) and analyzed these results against those of
DPA (Figures 3 and S10). In addition, various common cellular
63
fluorescence imaging. Fluorescence titrations have also been
performed to confirm the binding capability of DRHA with
DPA. The successive addition of a standard amount of DPA
showed enhancement in the emission band at 520 nm (λ =
ex
430 nm). The emission changes on the addition of DPA are
noticeable under UV light (inset Figure 2B). DRHA shows
evident emission spectra change on the addition of DPA.
Fluorescence intensities gradually increased with increasing
amounts of DPA. As shown in Figures 2C and S11, the
concentration of the DPA measurements can be linearly
correlated well with the fluorescence intensity values in the 0−9
μM concentration range with a correlation coefficient of
+
+
2+
2+
cations (Na , K , Ca , and Mg ) (Figure 3) were also tested
along with anions (Figure 2D). All or a number of this
66
interference may be the constituent of bacterial spores. This
study revealed that some biologically or structurally relevant
small molecule analytes have a slight or negligible effect on the
emission profile of DRHA as compared to DPA. It can be seen
that only DPA induces a prominent fluorescence enhancement
in the DRHA fluorescence profile at various concentrations,
concluding excellent selectivity of DRHA for DPA, which may
facilitate the recognition of this anthrax biomarker in a complex
environment.
0.9933. Using the linear response, a calibration curve can be
constructed, which can then be used to determine the amount
64
of DPA in an unknown sample quantitatively. Such a
sensitivity of the DPA sensor does not require any additional
calibration of the fluorescence intensity. The limit of detection
(
LOD) of this sensor can be defined as 3 × SD/m, where SD is
the standard deviation and m is the slope of the working curve.
The detection limit of DRHA for DPA is obtained to be 12 nM,
which is much less than the magnitude of an infectious dose of
The abovementioned results conclude that DRHA has good
practical applicability for environmental applications and has no
effect on the other environmental analytes present in water.
Finally, the workability of the proposed DRHA was tested for
the determination of DPA in real samples collected from
65
the spores for human beings (60 mM).
To evaluate the selectivity of the sensor, competitive binding
studies were carried out, where the host solution of DRHA (1
μM) was taken in different nine volumetric flasks, and then 1
G
DOI: 10.1021/acs.langmuir.8b00340
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Article
different sources (tap water, canal water, and rain water). To
establish the practical application, water samples were filtered
through a syringe filter to eliminate any biological impurity.
Now, the known concentrations of DPA (0, 20, and 40 nM)
have been pierced into the samples and tested against the
developed sensor. To check practical workability of the
synthesized sensor in real sample analysis, these samples were
subjected to analysis using proposed DRHA. The results are
shown in Table 1. It is clear from the results obtained that the
proposed assembly was able to determine the DPA
concentrations in these samples with a good precision value.
The ICT-based naphthalimide derivatives have been the
most accepted preference in chemosensors among all. The
reason behind its popularity is that it strongly absorbs and emits
at long wavelengths, which is highly desirable for sensing in
competitive media. Herein, a mechanism has been proposed
for binding of DRHA and DPA. The absorption and emission
changes are a result of CT interactions between the donor and
the acceptor. It is a well-known fact that the reaction of the
sensor with the interacting molecule switches on the ICT
process and induces an emission shift. Our approach to
fluorescence detection of DPA relies on ICT within the DRHA
platform. The electron-donating amino group on the organic
receptor affects both ICT and the emission profile, as making
the substituent more electron-deficient results in ICT-induced
hyperchromic shifts in emission maxima. Figure 4A demon-
strates the diagrammatic mechanism of recognition of DPA.
In support of this approach, ethidium bromide−DNA
interaction studies were carried out. Ethidium bromide
and the relative error was between 1.10 and 2.57%, which
indicated the acceptable accuracy and precision of the
developed assembly. Also, we performed the binding studies
of different batches of DRHA toward DPA. The obtained result
showed the same trend for all. It reveals that DRHA is a reliable
and reproducible sensing assembly for the detection of DPA.
Stability and Temperature Effects. The stability of
synthesized assembly was established under three conditions:
1
2
3
. Freeze−thaw stability: after going through three freeze−
thaw cycles (from −20 °C to room temperature)
. Short-term stability: at room temperature for the time of
regular work (at least 3 h)
. Long-term stability: in stock solutions at storage
conditions (−20 °C) for 1 month.
67
To ensure the reliability of results and handling/storing of
the synthesized assembly, stability studies were carried out. In
each situation, deviations from the original values were less than
±
5%. As shown in Tables S1 and S2, the synthesized assembly
was found to be stable for 3 h at ambient temperatures, after
three freeze−thaw cycles and storage for 1 month at −20 °C.
These results revealed that no spontaneous degradation was
found during the routine sample analysis and storage. Further,
absorption and emission spectra of DRHA were recorded after
3
h, freeze−thaw cycles, and after one month to check its short-
term and long-term stability. Both, the absorption and emission
spectra (Figure 5A,B) showed insignificant change in the
original values, which supported the stability of the system.
Comparison of the LOD of DRHA with Reported DPA
Sensors in the Literature. The performance and functioning
of the synthesized assembly for detection of DPA were also
compared with the previously reported lanthanide-based/
ratiometric fluorescent sensors. As is compared in Table 3,
the LOD acquired using synthesized assembly was comparable
(
EtBr) was added to a sample containing 10 μM of DRHA.
It was observed that the emission band at 545 nm remained
undisturbed, showing the insignificant effect of the addition of
EtBr on the emission spectra. In next step, to confirm the
interactions between DRHA and DPA, first DPA was added to
DRHA, and the solution was shaken properly. Then, EtBr
solution was added.
A new band was observed to appear near 610 nm typically
because of the EtBr/DNA complex formation (Figure 4B). It
supports the fact that the amino group of receptor 1 reacts with
DPA, leaving behind the DNA undisturbed. It reveals the
information that the amino group of receptor 1 reacts with
68−70
to some of the previously reported sensors,
a little more
while showing much lower value than
(Table 3). As is clear from the literature, the
26,71,72
than three of them,
64−66,72,73
others
LOD obtained (12 nM) by DRHA is lower than the
characteristic infectious dosage of B. anthracis spores (60
65
mM) for human beings. Moreover, the synthesized assembly
attains full advantage of water-soluble, good fluorescence
properties, including fast response, good precision and
accuracy, and stability over a good temperature range.
DPA, leaving the DNA undisturbed. A variation in IR spectra of
DRHA (in the absence and presence of DPA) near 1600 cm−1
also confirms the interactive nature of the amino group and
DPA (Figure 4C).
CONCLUSIONS
■
In conclusion, a naphthalimide-histidine receptor was synthe-
sized using the multistep reaction scheme. The fluorescent
organic receptor was then modified by the formation of its
DNA hybrid. Various techniques such as CD spectroscopy,
viscosity measurements, DNA denaturation experiments, DLS
studies, and time-resolved fluorescence studies have been done
to explore the DNA−receptor binding nature. The resulting
DRHA was used to detect DPA in an aqueous system with an
LOD of 12 nM. Response time studies exhibited quick binding
of the proposed assembly with DPA. Real sample analysis
confirmed the practical applicability of the probe for environ-
mental applications.
Statistical Analysis. Limit of Quantification (LOQ) and
LOD. The LOD and LOQ values were calculated from
duplicate measurements for each concentration of DPA. The
LOQ and the LOD for DPA were determined by multiplying
the standard error of the y-intercept by 10 and 3, respectively,
and dividing these values by the slope of the calibration curve.
The LOQ and LOD were determined to be 12 and 18 nM,
respectively.
Precision and Accuracy. Precision is a measure of the
reproducibility of the complete analytical process including
sample preparation and analysis under normal operating
conditions. The results concerning the accuracy and intra-
and interday precision of the method have been shown in Table
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.lang-
muir.8b00340.
2
for different concentration levels of DPA. Precision was
■
determined using the method to analyze a 20, 40, 60, and 80
nM DPA standard 10 times and then exhibiting the precision as
the relative standard deviation. The intra- and interday
precision values were less than 4.21 and 4.53%, respectively,
*
S
H
DOI: 10.1021/acs.langmuir.8b00340
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Spectroscopic data, FTIR, absorption spectra of receptor
Article
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biosensors for infectious agents: lesson from Bacillus anthracis
diagnostic sensors. Analyst 2010, 135, 1182−1190.
1
on the addition of DNA, DLS histogram, emission
spectra of DRHA upon the addition of various ligands,
linearity of a sensing mechanism for titration between
DRHA and DPA, and concentration of DPA added and
found after freeze−thaw cycles and at ambient temper-
atures (PDF)
(
15) Seo, Y.; Kim, J.-e.; Jeong, Y.; Lee, K. H.; Hwang, J.; Hong, J.;
Park, H.; Choi, J. Engineered nanoconstructs for the multiplexed and
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deMello, A. J.; Choo, J. Fast and sensitive detection of an anthrax
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AUTHOR INFORMATION
Corresponding Author
E-mail: nsingh@iitrpr.ac.in.
■
(
17) Okafor, A.; Uddin, M.; Ocando, J. E.; Jespersen, N.; Fan, J.;
*
Wang, E. Spectroscopic Study of Dipicolinic Acid Interaction with
Bovine Serum Albumin. J. Chem. Biochem 2016, 4, 23−46.
ORCID
(
18) Oh, W.-K.; Jeong, Y. S.; Song, J.; Jang, J. Fluorescent Europium-
Narinder Singh: 0000-0002-8794-8157
Notes
The authors declare no competing financial interest.
Modified Polymer Nanoparticles for Rapid and Sensitive Anthrax
Sensors. Biosens. Bioelectron 2011, 29, 172−177.
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ACKNOWLEDGMENTS
Light: Sci. Appl. 2013, 2, No. e62.
■
(20) Yilmaz, M. D.; Hsu, S.-H.; Reinhoudt, D. N.; Velders, A. H.;
M.V. and N.S. acknowledge the Science & Engineering
Research Board (SERB), New Delhi, for National Post
Doctoral Fellowship (PDF/2016/000159) and a research
project (EMR/2014/000613), respectively.
Huskens, J. Ratiometric Fluorescent Detection of an Anthrax
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DOI: 10.1021/acs.langmuir.8b00340
Langmuir XXXX, XXX, XXX−XXX