Analysis of Bisulfite Via a Nitro Derivative of Cyanine-3 (NCy3) in the Microfluidic Channel

Article abstract of DOI:10.1007/s10895-019-02394-0

NCy3, a derivative of Cyanine 3 with a nitro substituent, showed a high reactivity to bisulfite in aqueous media, instantly leading to ratiometric change of absorption spectra and significant fluorescence quenching. Applied in the microfluidic channel, NCy3 functionalize as a sensitive approach for quantitative detection of bisulfite, particularly for samples with a small volume.

Full text of DOI:10.1007/s10895-019-02394-0

Journal of Fluorescence
https://doi.org/10.1007/s10895-019-02394-0
SHORT COMMUNICATION
Analysis of Bisulfite Via a Nitro Derivative of Cyanine-3 (NCy3)
in the Microfluidic Channel
Haley A. Houtwed1 & Meng Xie1 & Aatiya Ahmad1 & Cody D. Masters1 & Melissa M. Davison1 & Kristy Kounovsky-Shafer1
&
Haishi Cao1
Received: 18 March 2019 /Accepted: 6 May 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
NCy3, a derivative of Cyanine 3 with a nitro substituent, showed a high reactivity to bisulfite in aqueous media, instantly leading
to ratiometric change of absorption spectra and significant fluorescence quenching. Applied in the microfluidic channel, NCy3
functionalize as a sensitive approach for quantitative detection of bisulfite, particularly for samples with a small volume.
.
.
.
Keywords Nitration Cyanine dye Bisulfite sensor Microfluidic channel
Introduction
small molecules in vitro and in vivo due to high selectivity,
sensitivity and real-time measurement [9]. Copious fluores-
cence approaches have been developed using different
photophysical mechanisms (e.g., photoinduced electron trans-
fer (PET), internal charge transfer (ICT), and (föster resonance
energy transfer (FRET)) for quantitative measurement of var-
ious bio-important molecules [10]. However, the sensitivity
and selectivity are always a challenge for fluorescence sensing
approaches, particularly in biosamples [11].
Microfluidic devices offer scientists many benefits com-
pared to larger scale systems. One benefit of using
microchannels is the small volume needed to analyze a
sample. Depending on the size of the channel and the num-
ber of channels, only femtoliters of the solution are re-
quired, which is cost-effective and beneficial for experi-
ments with limited samples [12]. Additionally, microfluidics
lends well to multiplexing, so a scientist can run multiple
tests on a chip. Finally, microfluidics has a faster reaction
time, portability, and enhanced sensitivity compared to
large-scale systems [13]. Many systems already use
microfluidics such as sequencing, detection of metabolites,
physical DNA mapping, 3D cell culture, and optofluidics
due to the aforementioned benefits [12–15].
Preservative and antimicrobial reagent against oxidation
and spoilage during food processing and wine fermenta-
tion [1], and is also added to dried fruit and potato prod-
ucts for long term storage [2]. Based on the report from
the World Health Organization (WHO), the daily intake
limit of sulfite is 0.7 mg/Kg [3]. High doses of sulfide
shows a highly biological toxicity, causing disease and
allergic reaction [4]. Recent studies reveal that bisulfite
is correlated to lung cancer, cardiovascular disease and
many neurological disorders [5]. However, bisulfite also
exists naturally in the human body. In living cells, sulfite
and bisulfite can be produced in aqueous media at phys-
iological condition by endogenous sulfur dioxide (SO2),
which is generated from the oxidation of H2S or sulfur
containing amino acids by reactive oxygen species
(ROS) [6]. Thus, the cellular level of bisulfite is able
to collaterally reflect the local reducing environment in
living cells [7]. Therefore, developing an efficient ap-
proach for rapid detection of sulfite is highly desired [8].
In past years, the fluorescence chemosensor has attracted a
substantial amount of attention as a simple tool used to detect
In our group, we recently developed a reaction-based
fluorescence sensor based on Cyanine 3 dye, which was
−
able to rapidly react with HSO3 based on a nucleophil-
−
* Haishi Cao
ic addition mechanism. In the presence of HSO3
,
Caoh1@unk.edu
NCy3 showed a ratiometric change of absorption spec-
tra and a strong fluorescence quenching within 3 min at
25 °C, indicating a high sensitivity and selectivity.
1
Department of Chemistry, University of Nebraska-Kearney,
Kearney, NE 68849, USA

J Fluoresc
Table 1 The photophysical properties of NCy3 [17]
Methods
Solvent
λab(nm)
λab(em)
ε(cm−1M−1
)
Φ
Apparatus
DMSO
Acetone
MeOH
THF
578
566
564
578
576
567
601
583
581
595
592
581
27,800
32,600
15,200
22,900
24,700
34,900
0.190
0.140
0.148
0.156
0.108
0.160
All reagents used for synthesis and measurements were pur-
chased from Sigma-Aldrich (MO, USA), Fisher Scientific
(USA), TCI (USA), Alfa Aesar (USA) and Acros Organics
(USA) in analytical grade and were used as received, unless
otherwise stated. Absorbance spectra were collected by Cary
Series Uv-vis Spectrophotometer (Agilent Technologies).
Fluorescence measurements were all performed by using a
FluoroMax-4 Spectrofluorometer (Horiba Jobin Yvon, USA).
All of fluorescence spectra were recorded in a 1 cm quartz cu-
vette. The excitation and emission slits were set at 2 nm. 1H and
13C NMR spectra were recorded on (1H 300 MHz, 13C 75 MHz)
Bruker 300 Ultra-Shield spectrometer at room temperature. The
HRMS data was collected in the Nebraska Center of Mass
Spectrometry at University of Nebraska-Lincoln by using GCT
Mass Spectrometer (Water, USA).
EtOAc
CH2CI2
NCy3 was also applied to a microfluidic device to mea-
sure the fluorescence intensity of samples with a small
volume, in which a similar result was obtained, indicat-
ing NCy3 could be used as an approach for quantitative
detection of sulfite with a high affinity and selectivity in
the aqueous media.
Synthesis
2,3,3-trimethyl-5-nitro-3H-indole (1). 2,3,3-trimethylindole
(3.18 g, 0.02 mol) was added into concentrated H2SO4
(75 mL) containing HNO3 (1.70 g, 0.02 mol) within 1 h and
was incubated for 0.5 h at 0 °C [16]. After reaction, the mix-
ture was poured into ice (1000 mL) to collect the precipatate
by suction filtration. The solid was recrystallized in CH2Cl2 to
yiled an organge ctrystall as the product (3.5 g, 91%). 1H
NMR (300 MHz, CDCl3) δ: 1.4 (s, 6H), 2.4 (s, 3H), 7.6 (d,
J = 8.3 Hz, 1H), 8.2 (s, 1H), 8.3 (d, J = 8.2 Hz, 1H). 13C NMR
(75 MHz, CDCl3) δ: 16.1, 22.9, 54.6, 117.3, 120.3, 124.8,
145.8, 147.0, 159.3, 194.3.
3H-indol-1-ium (NCy3). Compound 2 (0.218 g, 0.001 mol)
and triethyl orthoformate (0.593 g, 0.004 mol) were heated in
acetic acid/acetic anhydride (1:1, 5 mL) at 100 °C for 12 h.
After cooling to room temperature, the reaction mixture was
purified by column chromatography (silica, 220–400 mesh).
Ethyl acetate and methanol (4:1) were used as the elution
solvents. A dard red solid (NCy3) was obtained as the product
1
(0.170 g, 52%). H-NMR (400 MHz, DMSO-D6) δ: 1.8 (s,
12H), 3.7 (s, 6H), 6.7 (d, J = 13.5 Hz, 2H), 7.7 (d, J = 8.4 Hz,
2H), 7.7 (t, J = 5.7 Hz, 2H), 8.3–8.4 (m, 3H), 8.6 (s, 2H). 13C-
NMR (75 MHz, DMSO-D6) δ: 27.4, 32.7, 49.6, 106.2, 112.6,
118.8, 126.1, 142.4, 145.0, 148.3, 152.2, 177.3. TOF EI+: M+
m/z 447.2027 (calcd.), 446.5980(found).
1,2,3,3-tetramethyl-5-nitro-3H-indol-1-ium (2). A mixture
of 1 (0.40 g, 0.0020 mol) and CH3I (0.33 g, 0.0024 mol) were
heated to 90 °C in CCl4 (4 mL) for 15 h in a sealed reaction
tube (50 mL). After cooling to room temperature, the reaction
mixture was filtrated to yield a brown yellow solid as the pure
Microfluidic Methods
1
product without any further purification (0.41 g, 95%). H-
Master and Replica Fabrication
NMR (400 MHz, CDCl3) δ: 1.4 (s, 6H), 3.2 (s, 3H), 4.2 (d,
J = 10.5 Hz, 2H), 6.5 (d, J = 6.7 Hz, 1H), 8.0 (s, 1H), 8.2 (d,
J = 8.2 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ: 29.4, 29.6,
43.5, 80.2, 105.5, 118.5, 126.7, 138.5, 139.6, 152.1, 161.3.
1,3,3-trimethyl-5-nitro-2-((E)-3-((E)-1,3,3-trimethyl-5-
nitroindolin-2-ylidene)prop-1-en-1-yl)-5,6,7,7a-tetrahydro-
SU8 microchannel masters were fabricated by photolithography
by spincoating SU8 2005 (5.0 μm, MicroChem) onto a silicon
wafer and exposing it with UV-light. Replicas of the masters
were created using soft lithography. PDMS (Sylgard 184) was
mixed (10:1 ratio of pre-polymer to Platinum catalyst) for 15 min

J Fluoresc
−
Fig. 1 The spectra change of NCy3 (1.0 × 10−5 M) with addition of HSO3 (0–65 μM) in DMSO/pH 7.4 buffer (1:1) at 25 °C. a The absorption
decreased at 574 nm and increased at 455 nm; b The fluorescence emission at 594 nm was gradually quenched (λex = 574 nm)
with a hand held mixer (Kitchen Aid 9 speed mixer), poured onto
the silicon master, and placed in a 65 °C oven overnight. The
PDMS replica was removed from the master and then made
hydrophilic with oxygen plasma treatment (Diener Zepto
System, 36 s O2 plasma, 0.50 mbar pressure, 15% power) and
stored in distilled water until ready to use.
TE2000-S microscope. A Brightline 4040B filter set was used
with X-Cite 120 Fluorescence Illumination light source to
excite the dye and was captured with the NIS Elements D
capture software. ImageJ was used to analyze images to com-
pile the 2D montage and determined the fluorescence intensity
for each image.5
Imaging of Dye in Microchannels
Results and Discussion
The PDMS device was mounted onto coverslips and attached
to a holder with candle wax. Dye (1 μL, 1 × 10−3 M) and 3 μl
The absorption and emission spectra of NCy3 were collected
in the different media, in which the maximum absorption and
emission were observed in the range of 564–578 nm and 581–
601 nm respectively (Table 1). NCy3 showed the highest
quantum yield (0.190) in DMSO and the lowest quantum
−
distilled water or 3 μl HSO3 (1 × 10−3 M) was mixed and
loaded into microchannels via capillary action. The dynamics
−
of the dye in the presence of water or HSO3 was imaged
using a CoolSNAP camera and coupled to a Nikon Eclipse
Fig. 2 The time-dependent spectrum change: (a) absorption at 574 nm, (b) absorption at 455 nm, and (c) emission at 594 nm of NCy3 (1.0 × 10−5 M)
with different amount of HSO3− (5 μM, 10 μM, 20 μM, and 30 μM) in DMSO/pH 7.4 buffer (1:1) at 25 °C

J Fluoresc
Fig. 3 The absorption responses (a) and fluorescence emission responses (b) of NCy3 (1.0 × 10−5 M) toward HSO4−, S2O32−, C2O42−, Cys, GSH,
ascorbic acid, H2S and HSO3− (30 μM) after Incubation for 5 min in DMSO/pH 7.4 buffer (1:1) at 25 °C (λex = 574 nm)
yield (0.108) in ethyl acetate. Based on the Consideration of
solubility and photophysical properties, we chose a mixture of
DMSO and H2O (5:5, v/v) solution containing 20 mM phos-
instant color change, red to light yellow, was also observed
by the naked eye. The maximum spectrum change was
achieved when the concentration of HSO3 reached 30 μM,
−
−
phate buffer at pH 7.4 for NCy3 to detect HSO3 .
suggesting the accomplishment of the reaction. In the emis-
−
−
As a nucleophile, HSO3 may react with NCy3 via a nu-
sion spectra, HSO3 led to a gradual fluorescence quench at
cleophilic addition reaction, which interrupts the conjugation
of NCy3 and consequently change the photophyscial proper-
ties. We first investigated the absorption and emission spectra
of NCy3 in the presence of HSO3− in the DMSO/buffer solu-
tion. With addition of HSO3− to NCy3, the absorption spectra
rapidly showed a ratiometric signal, a dramatic decrease at
574 nm and an increase at 455 nm, indicating a significant
structure change of NCy3 (Fig. 1).
597 nm, and the maximum quench (92%) was observed with
the addition of 30 μM HSO3 , which was consistent with the
−
change of absorption spectra. The detection limit (3δ/k) was
calculated to be 0.086 nM based fluorescence titration at
597 nm [18].
For the reaction-based sensor, the response time is a critical
factor that affects the sensing process. The chemical reaction-
based signal significantly improves the sensor selectivity, but
limits applications if the reaction rate is too slow. Therefore,
the time-dependent spectrum change was investigated after
In the absorption spectra,a remarkable blue-shift (119 nm)
was detected due to the interruption of π-conjugation. The
Fig. 4 The spectra change of NCy3 (1.0 × 10−5 M) incubating with
different amount of H2S (10 μM, 50 μM, 100 μM, and 150 μM) in
DMSO/pH 7.4 buffer (1:1) at 25 °C in 45 min: (a) the absorption
change at 574 nm, (b) the absorption change at 455 nm, and (c) the
fluorescence change at 594 nm (λex = 574 nm)

J Fluoresc
Fig. 5 a NCy3 reacted with H2S and HSO3− based on a nucleophilic addition mechanism. b The fluorescence intensity change of NCy3 at 594 nm in
different media with a pH 3.0–11.0 at 25 °C in (λex = 574 nm)
−
incubation with different amount of HSO3 (5 μM, 10 μM,
20 μM, and 30 μM) to NCy3 at 25 °C in the DMSO/buffer
media. A change of absorption at 574 nm and 455 nm were
measured as well as the emission at 597 nm. A nucleophilic
reaction triggered a spectrum change instantly. After incubat-
ing HSO3− (5 μM) with NCy3 at 25 °C, both absorption and
emission spectra gradually changed over the course of 25 min.
biothiols, reducing reagents, and sulfur-containing ions
(30 μM) were incubated with NCy3 (1.0 × 10−5 M) for
5 min in a DMSO/Buffer media at 25 °C. Afterwards, the
absorption and emission spectra were measured. As shown
−
2−
in the Fig. 3, all of interfering species (HSO4 , S2O3
,
C2O42−, cysteine, glutathione, ascorbic acid, and H2S) showed
just a slight change (i.e., around 10%) in both absorption and
the emission spectra. However, during incubation with
−
However, a high concentration of HSO3 (30 μM) led to a
−
rapid spectra change and the maximum change was observed
within 5 min, which indicated a high reaction rate based on
our data for our reaction-based sensor (Fig. 2).
High affinity for a target molecule is another important
factor to evaluate a sensor, and is one of the main challenges
for sensor designs. To examine the selectivity of NCy3 to-
wards different molecules, various analytes, including
HSO3 , NCy3 rapidly displayed a ratiometric signal at
455 nm/575 nm in the absorption spectra as well as a signif-
icant quenching at 597 nm in the fluorescence spectra, dem-
−
onstrating that NCy3 is very sensitive to HSO3 , allowing
NCy3 to be used as a approach with multiple spectroscopic
−
signal channels for rapid detection of HSO3 . The interruption
−
of the conjugation of NCy3 trigged by HSO3 led to an
Fig. 6 After incubation Cy3 (1.0 × 10−5 M) with HSO4−, S2O32−, C2O4
addition reaction observed; (b) absorption spectra and (c) fluorescence
spectra did not show a significant change
2
−, Cys, GSH, ascorbic acid, H2S and HSO3 (150 μM) for 30 min in
−
DMSO/pH 7.4 buffer (1:1) at 25 °C (λex = 574 nm), (a) no nucleophilic

J Fluoresc
−
Fig. 7 a Intensity of the dye with water (red circles) or HSO3 (black
compiled into a movie. A 2D slice was taken from each image at the same
location and compiled to create the 2D montage (1 min per slice). The
distance (x) of the slices of the image was in μm
squares) in 55 μm wide × 5.0 μm high microchannels was normalized
and plotted against time. (B) and (C) Dynamics of the dye in the presence
of water (b) or HSO3−(C) was imaged in microchannels for 10 min and
obvious color change from light pink to yellow, which can be
easily observed by naked eye, making the detection process
very convenient.
dye (Cy3) without nitro groups was synthesized for a compar-
ison. Compared to NCy3, Cy3 showed a shorter absorption
and emission at 546 nm and 565 nm respectively in the
DMSO/pH 7.4 buffer (1:1) at 25 °C. Since NCy3 showed a
significant spectroscopic change in the presence of HSO3− and
H2S in high concentration, the same condition was applied to
Cy3. However, neither absorption nor emission displayed a
notable change. Moreover, high concentration (150 μM) of
As a nucleophile, H2S did not show a high reaction rate with
NCy3 at low concentration. However, with increasing concen-
tration and incubation time, NCy3 gave a significant response
to H2S. As shown in the Fig. 4, both the absorption and emis-
sion spectra steadily changed within 45 min at 25 °C when the
H2S level was less than 100 μM, indicating a slow reaction
process. When increasing the level of H2S to 150 μM, the
spectra displayed a relatively quick change, and plateaued after
a 30-min incubation with NCy3, suggesting a complete reac-
tion. Based on the previous data, NCy3 showed a potential to
detect H2S with a high concentration. Besides H2S, other spe-
−
analytes (HSO4−, S2O32−, C2O42−, Cys, GSH, ascorbic acid,
H2S, and HSO3 ) along with longer incubation time (30 min)
with Cy3 could not produce any significant spectroscopic
change, revealing that no nucleophilic addition reaction oc-
curred for Cy3 (Fig. 6). The nucleophilic addition reaction is
well established for α,β-unsaturated compounds. As an
electron-withdrawing group, the nitro group on NCy3 played
a critical role in significantly boosting the addition reaction.
Moreover, the HRMS data indicated that a reduction reaction
followed the nucleophilic addition reaction in the present of
−
cies (HSO4 , S2O32−, C2O42−, Cys, GSH, and ascorbic acid)
did not cause significant spectroscopic change even at high
concentration and/or longer incubation time with NCy3.
According to previous work, the nitro group on the aromat-
ic ring (1,8-naphthalimide) can be reduced by H2S and con-
sequently lead to photophysical property change, which has
been intensively used as a sensing strategy for the detection of
H2S [19]. Thus, NCy3 was incubated with different reducing
−
HSO3 , which thoroughly explained the significant spectra
change for absorption and emission.
Because NCy3 was ultra-sensitive, ratiometric, instant, and
multiple channel signal sensing abilities, NCy3 was applied to
−
−
reagents (H2S, GSH, ascorbic acid, and HSO3 ) to investigate
develop a microfluidic device for analysis of HSO3 in very
the reduction of nitro group. However, no reduction reaction
was observed in the presence of any these reducing reagents.
Only HSO3− and H2S led to a nucleophilic addition reaction,
and HSO3− gave a higher reaction rate (Fig. 5a). Moreover, the
pH effect in the range of pH 3–11 was examined for NCy3 in
different DMSO/buffer media. At low pH range (3.0–8.0),
NCy3 showed a strong fluorescence emission at 597 nm.
With an increase of pH, the fluorescence was quenched rap-
idly. Although the fluorescence intensity of NCy3 was signif-
icantly influenced by pH value, no notable change was ob-
served in the physiological pH range (pH 7.0–8.0) (Fig. 5b).
To understand the nucleophilic addition reaction between
NCy3 and nucleophiles (e.g., HSO3− and H2S), the Cyanine 3
small volume samples. A PDMS device was mounted onto a
coverslip and attached to a 3D printed PLA holder with candle
wax [20]. Then, dye (1 μl, 1.0 × 10−3 M) and 3 μl distilled
water or 3 μl HSO3 (1.0 × 10−3 M) was mixed and loaded
−
into microchannels via capillary action. The dynamics of the
−
dye in the presence of water or HSO3 was imaged using a
CoolSNAP camera and coupled to a Nikon Eclipse TE2000-S
−
microscope. As shown in Fig. 7, with the incubation of HSO3
with NCy3 in the microchannel, a fluorescence quenching
was observed within 10 min, indicating that NCy3 was able
to produce a reliable signal in a microfluidic device for detec-
−
tion of HSO3 . As a negative control, addition of H2O did not
led to a significant change in fluorescence.

J Fluoresc
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Conclusions
In summary, a fluorescent probe (NCy3) was synthesized for
detection of HSO3 via a nucleophilic addition reaction in
−
aqueous media. NCy3 demonstrated a rapid ratiometric sens-
ing for HSO3− with high selectivity and sensitivity. This probe
provided multiple signals, including absorption spectra, emis-
sion spectra, and colormetric signal, to quantitatively measure
−
HSO3 . In addition, NCy3 was successfully employed in de-
veloping a microfluidic device to measure trace amounts of
HSO3− in samples with a small volume, which could be very
useful for biosamples or environmental monitoring.
Acknowledgments Thanks Dr. Brandon Luedtke for help with the Nikon
microscope. Financial support was provided by grants from the National
Center for Research Resources (NCRR; 5P20RR016469) and the
National Institute for General Medical Science (NIGMS; INBRE-
8P20GM103427), a component of the National Institutes of Health
(NIH) and Nebraska Research Initiative for the purchase of the Nikon
microscope.
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