A new ratiometric fluorescence assay based on resonance energy transfer between biomass quantum dots and organic dye for the detection of sulfur dioxide derivatives

Article abstract of DOI:10.1039/c9ra09437g

Sulfur dioxide (SO₂) is considered as the fourth gas signal molecule after nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H₂S). It plays important roles in several physiological processes. Therefore, the design and syn

Full text of DOI:10.1039/c9ra09437g

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A new ratiometric fluorescence assay based on
resonance energy transfer between biomass
quantum dots and organic dye for the detection of
sulfur dioxide derivatives†
Cite this: RSC Adv., 2019, 9, 41955
ab
a
*
Jingjin Zhao,
and Yi-Ming Liu
‡
Yao Peng,‡a Keqin Yang,a Yunyun Chen,a Shulin Zhao
*
b
Sulfur dioxide (SO2) is considered as the fourth gas signal molecule after nitric oxide (NO), carbon monoxide
(CO) and hydrogen sulfide (H2S). It plays important roles in several physiological processes. Therefore, the
design and synthesis of nanoprobes for the detection of SO2 derivatives in cells is of great significance.
Herein, we report a new ratiometric fluorescence nanoprobe based on resonance energy transfer (RET)
between biomass quantum dots (BQDs) and organic dye (DMI) for the detection of SO2 derivatives. The
Received 13th November 2019
Accepted 10th December 2019
ꢀ
proposed ratiometric fluorescence assay allows the determination of HSO3 in the range of 1.0 to 225
mM with a detection limit of 0.5 mM. Importantly, the proposed ratiometric fluorescence nanoprobe
DOI: 10.1039/c9ra09437g
ꢀ
exhibits a high photostability and good selectivity for HSO3 over other chemical species including H2S
rsc.li/rsc-advances
and biological mercaptans. Quantitation of HSO3ꢀ in cell lysates by using the nanoprobe is demonstrated.
(HPLC),10 capillary electrophoresis,11 etc. These methods have
limitations such as poor reproducibility, un-satisfactory assay
1. Introduction
sensitivity, or cumbersome operation. In recent years, uores-
cence based methods have received attention due to their high
sensitivity, good selectivity, ꢀand short analysis time. Several
uorescent probes for HSO3 detection were developed based
on nucleophilic reactions with aldehydes, levulinic acid
cracking and coordination interactions.12–18 However, these
uorescent probes are susceptible to interference from endog-
enous compounds such as biological mercaptans, proteases
and esterases. In addition, it's well known that uorescence
assays based on changes in single-wavelength uorescence
intensity (increase or decrease) are affected by probe concen-
tration, instrument stability, and background interference of
the test system.19–21
Ratiometric uorescence assay is more accurate than
single-wavelength uorescence intensity detection because it
eliminates most of the interference caused by light bleaching,
instrument instability, and sample matrix background by self-
calibration on two emission bands. In recent years, many ratio
uorescence probes were developed for SO2 assay.22–31
However, most of these probes were designed based on the
mechanism of intramolecular charge transfer (ICT). Such
probes have shortcomings such as low uorescence resolu-
tion, and being susceptible to the inuence from other bio-
logical mercaptan. On the other hand, these organic small
molecular uorescent probes are prone to photobleaching
when exposed to light, so their optical stability is not very
good. Therefore, it is highly signicant to develop stable,
Sulfur dioxide (SO2) is an irritant gas and a major atmospheric
2ꢀ
ꢀ
pollutant. It is usually in the form of SO3 and HSO3 in
solution.1,2 In vivo, SO2 is considered as the fourth gas signal
molecule aer nitric oxide (NO), carbon monoxide (CO) and
hydrogen sulde (H2S).3 SO2 can be produced in cytoplasm and
mitochondria by enzymatic catalysis of sulfur-containing amino
acids and oxidation of H2S.4 Endogenous SO2 has unique bio-
logical activity and plays an important role in a series of phys-
iological and pathological processes, such as improving
antioxidant capacity, regulating vascular structure and func-
tion, etc.5,6 On the other hand, studies have shown that
abnormal production of endogenous SO2 derivatives is associ-
ated with certain diseases such as neurological disorders7 and
lung cancer.8 Therefore, the development of analytical methods
with good repeatability and sensitivity for the detection of SO2
derivatives in cells is of great signicance.
Current methods for the detection of HSO3ꢀ (a major form of
SO2 existing in living systems) include electrochemical detec-
tion method,9 high performance liquid chromatography
aState Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, Guangxi Normal University, Guilin, 541004, China. E-mail:
zhaoshulin001@163.com
bDepartment of Chemistry and Biochemistry, Jackson State University, 1400 Lynch St.,
Jackson, MS 39217, USA. E-mail: yiming.liu@msjsu.edu
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra09437g
‡ These two authors contributed equally to this work.
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sensitive and specic ratiometric uorescence assay for SO2 (Shanghai, China). Dulbecco's Modied Eagle Medium (DMEM)
derivatives. was purchased from Thermo Fisher instrument Co., Ltd. (Suz-
Biomass quantum dots (BQDs) are uorescent carbon based hou, China). Trypsin digestive uid, fetal bovine serum (FBS),
nanomaterials prepared from biomass. They are easy to be dimethyl sulfoxide (DMSO) and penicillin–streptomycin were
prepared, easy to be functionalized, with good photostability, purchased from Sigma-Aldrich Company (Saint Louis, MO,
low cytotoxicity and good biocompatibility. Moreover, BQDs USA). The cell lines used in the study: HepG2, MCF-7 and 7702
with different optical properties can be prepared from different were purchased from the Cell Bank of the typical Culture
biomass.32–34 In this work, we prepared a BQDs with green Storage Committee of the Chinese Academy of Sciences/Cell
uorescence emission from corn, and developed a ratiometric Resource Center of the Shanghai Institute of Life Sciences of
uorescence assay based on resonance energy transfer (RET) the Chinese Academy of Sciences (Shanghai, China). Dialysis
between BQDs and organic dye for the detection of sulfur bag (MWCO ¼ 1 kD, pore size: ca.1.0 nm) purchased from
dioxide derivatives (HSO3ꢀ). The principle of the assay is out- Shengong Bioengineering Co., Ltd. (Shanghai, China). All the
lined in Scheme 1. BQDs were prepared from corn by one-step other chemical reagents in the experiment were analysis pure,
hydrothermal method, and (E)-2-(4-(dimethylamino) styrene)- and the water used in the experiment was 18.2 MU cm ultrapure
1,3,3-trimethyl-3H-indole-1-iodide (DMI) was synthesized by water.
two-step nucleophilic reaction. Both BQDs and DMI obtained
showed the uorescence with maximum emission wavelengths 2.2 Apparatus
at 518 nm and 595 nm, respectively. DMI had maximum
absorption at 549 nm, which was overlapped with the emission
spectrum of BQDs. When the two co-exist, uorescence reso-
nance energy transfer (FRET) occur, which results in the uo-
The Cary Eclipse uorescence spectrometer (Agilent Technolo-
gies, Santa Clara, CA, USA) was used for recording the uores-
cence spectrum. The Cary-60 UV-vis spectrophotometer (Agilent
Technologies) was used for recording the UV-vis absorption
rescence intensity of BQDs at 518 nm decreases while that of
spectrum. The PerkinElmer Fourier transform infrared (FTIR)
DMI at 595 nm increases. In the presence of HSO3ꢀ, absorption
spectrometer (PerkinElmer Inc./Thermo Fisher Scientic, Wal-
ꢀ
of DMI at 549 nm is reduced because HSO3 reacts with DMI
tham, MA, USA) was used for characterization of the BQDs
breaking up the conjugation of the double bonds in DMI, thus
surface groups. The Rigaku X-ray powder diffractometer
(Rigaku Corp., Tokyo, Japan) was used for X-ray diffraction
(XRD) analysis. The Philips transmission electron microscope
diminishing FRET efficiency. As a result, uorescence emission
by DMI is decreased at 595 nm, while uorescence of BQDs is
enhanced at 518 nm. Fluorescence intensity ratio of the system
(Philips, Eindhoven, Netherlands) was used to characterize the
at 518 nm and 595 nmꢀ(F518/F595) is linearly proportional to the
particle size range of the BQDs. The ESCALAB™ X-ray photo-
concentration of HSO3 within a certain range. Based on this
electron spectrometer (Thermo Fisher, Waltham, MA, USA) was
workiꢀng principle, a new ratiometric uorescence method for
used for elemental analysis of BQDs (XPS). Polytetrauoro-
HSO3 detection can be established.
ethylene reactor (50 mL, Jinan Henghua technology Co., Ltd.)
was used for the synthesis of BQDs. FS-150N ultrasonic
processor (Shanghai Biogen ultrasonic instrument Co., Ltd.)
was used to prepare cell lysates.
2. Experimental section
2.1 Materials and reagents
2.3 Synthesis of BQDs
Corn was bought from a local vegetable market (Guilin, China).
2,3,3-trimethyl-3H-indole, 4-diaminobenzaldehyde, acetoni-
trile, anhydrous ethanol, iodine methane, NaNO2, KBr, NaCl,
KI, Na2HPO4, NaNO3, NaHCO3, Na2CO3, Na2S, CH3COONa,
cystine (Cys), glutathione (GSH), H2O2, ascorbic acid (AA) and
NaHSO3 were purchased from Aladdin reagent company
Corn was washed with tap water and ultrapure water, and blow
dry the surface. Then, 50 g corn was placed into a mortar, fully
grind it and transfer it to the high-pressure reactor lined with
polytetrauoroethylene (volume: 50 mL), 20 mL ultrapure water
was added into the high-pressure reactor. Place the high-
pressure reactor in the SZCL-3B digital display intelligent
temperature control magnetic stirrer for stirring and heating to
ꢁ
260 C for 10 h for reaction, turn off the heating and continue
stirring, and let it cool naturally to room temperature. The
mixture in the reactor was ltered with 0.22 mm drainage
membrane, and the ltrate was transferred to dialysis bag
(MWCO ¼ 1 kD, pore size: ca. 1.0 nm). Aer dialysis in ultrapure
water for 12 h, green uorescent BQDs were obtained.
2.4 Synthesis of DMI
Typically, 3.2 g (20 mmol) 2,3,3-trimethyl-3H-indole and 5.68 g
(40 mmol) iodine–methane were mixed and dissolved in 10 mL
acetonitrile solution. The mixture solution was heated to 60 ꢁC
under the protection of argon, and stirred for 11 h under the
Scheme 1 Schematic illustration of the proposed ratiometric fluo-
rescence assay based on FRET between BQDs and organic dye (DMI)
for the detection of SO2 derivatives.
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reux of cooling water. The heating was stopped, and aer placed for 10 min at room temperature. Then, the uorescence
natural cooling to room temperature, the light pink precipita- spectrum of the solution was measured on a uorescence
tion was obtained. Aer ltration with qualitative lter paper, spectrometer with 446 nm excitation.
the precipitation was washed with ethyl acetate three times. The
precipitation was then dried in a vacuum drying oven to obtain
a light pink powder (5.8 g).
3. Results and discussion
3.1 Characterization of BCDs
The light pink powder 0.150 g (0.50 mmol) and 0.12 g (0.75
mmol) 4-(dimethylamine) benzaldehyde were mixed and dis-
solved in 10 mL ethanol, and placed in a three-necked ask,
The morphology, surface functional groups, structure and
composition of the BQDs were investigated by transmission
electron microscope (TEM), X-ray diffraction (XRD), Fourier
transform infrared spectroscopy (FTIR) and X-ray photo-electric
spectrometry (XPS), respectively. TEM of BQDs is shown in
Fig. 1a. It can be seen from the gure that the prepared BQDs
are spherical particles and evenly dispersed, indicating they are
water-soluble. The particle size distribution is within the scope
of 2.0 to 5.5 nm, average particle size is 3.5 nm (Fig. 1b), and
high resolution TEM (HRTEM) (inset in Fig. 1a) shown a clear
lattice stripes, lattice constant is 0.315 nm. This corresponds to
graphite material (002), indicating that the BQDs consist of
graphite-like structures. The crystal surface constant of BQDs is
characterized by XRD, and the results are shown in Fig. S1
(ESI).† It can be seen from the gure that the BQDs show a wide
diffraction peak at the position of 2q ¼ 23.43ꢁ. This diffraction
peak corresponds to the amorphous carbon structure crystal
plane (002).35 Functional groups of BQDs surface are charac-
terized by FTIR, and the results are shown in Fig. S2 (ESI).† The
peak at 1027 cmꢀ1 corresponds to the stretching vibration peak
of C–O bond, the peak at 1652 cmꢀ1 corresponds to the
stretching vibration peak of C]O, the peak at 2939 cmꢀ1
belongs to the C–H bond, and the peak at 3367 cmꢀ1 corre-
sponds to the stretching vibration peak of O–H/N–H. The
results show that on the surface of BQDs hydrophilic groups
such as –COOH and –OH are present which lands BQDs with
a good water solubility. The element composition and surface
bonding of BQDs were characterized by XPS. The results indi-
cated that the BQDs were composed of ve elements C, N, O, S
and P. The composition was: C 64.44%, N 11.80%, O 23.15%, S
ꢁ
heated to 180 C with argon protection, and the cooling water
was reated for 4.5 h to obtain the red solution. The solvent was
removed from the rotary evaporator, and a puried organic
small molecule compound was obtained by column chroma-
tography (CH2Cl2/C2H5OH): (E)-2-(4-(dimethylamino) styryl)-
1
1,3,3-trimethyl-3H-indol-1-ium iodide (DMI, 0.194 g). H NMR
(400 MHz, CDCl3) d 8.14 (d, J ¼ 15.2 Hz, 2H), 7.49–7.43 (m, 3H),
7.34–7.30 (m, 1H), 6.82 (d, J ¼ 8.8 Hz, 2H), 5.32 (s, 1H), 4.17 (s,
3H), 3.20 (s, 6H), 1.99 (s, 1H), 1.80 (s, 6H); 13C NMR (100 MHz,
CDCl3) d 179.0, 155.1, 154.8, 141.8, 141.6, 129.0, 127.6, 122.3,
122.2, 112.7, 112.5, 104.6, 53.4, 50.8, 40.4, 35.1, 27.5. The
synthetic rout is shown in the following gure:
2.5 Preparation of cells lysates
Cells used in the study were HepG2, MCF-7 and 7702 cells. The
cell lines were cultured in a DMEM culture medium containing
10% fetal bovine serum, 10 U mLꢀ1 penicillin, 10 g mLꢀ1
streptomycin, and incubated at 37 ꢁC for 24 h in a 5% CO2
atmosphere. Aer digesting with trypsin, the culture medium
was removed by centrifugation, and the cells were dispersed in
the PBS buffer. This procedure was repeated three times. Then,
the cells were resuspended in 1ꢂ PBS buffer. A 3 mL volume of
above cell suspension was added into the centrifuge tube, and
centrifuging for 5 min at a speed of 1000 rpm. Then, the PBS
solution was abandoned, 200 mL 1ꢂ PBS buffer solutions were
used to suspend the cells with a cell density of about 1.43 ꢂ 106
cells per mL, and transfer into a 1.5 mL centrifuge tube. The
centrifugal tube was put in ice water bath on the ultrasonic
instrument, with 80 W ultrasonic power ultrasonic broken for
20 min. Aer ultrasonic fragmentation, ultraltration
membranes with a molecular weight cutoff of 10 kDa were used
to remove large molecular weight proteins and extract the
ltrate was retained for analysis.
ꢀ
2.6 Determination of HSO3 in cells lysate
Typically, 20 mL cell lysate was added into 150 mL BQDs-DMI
mixture solution (1ꢂ PBS : C2H5OH ¼ 7 : 3, concentration of
DMI is 80 mM), and added a certain amount of ultrapure water
Fig. 1 Characterization of BQDs prepared: (a) TEM and HRTEM
images; (b) particle size distribution; (c) XPS spectrum; (d) UV-vis
to a nal volume of 200 mL. Aer mixing well, the solution was absorption and fluorescence spectra.
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0.268% and P 0.332%. According to the overall distribution
diagram of element analysis (Fig. 1c), the C 1s characteristic
peak appeared at the position of 284.9 eV, the N 1s character-
istic peak appeared at the position of 399.7 eV, the O 1s char-
acteristic peak appeared at the position of 531.5 eV, and the S 2p
and P 2p characteristic peaks appeared at the position of
163.5 eV and 134.2 eV respectively. As can be seen from the
high-resolution spectrum of C 1s (Fig. S3a, ESI†), the signal
peak appears at 287.8 eV, 286.3 eV, 285.7 eV, 285 eV and
284.3 eV of C 1s, in which 287.8 eV is C]O bond. 286.3 eV is
carbon sp3 hybrid C–O bond; 285.7 eV is carbon sp3 hybrid C–N
bond; 285 eV is carbon sp2 hybrid C]C bond; 284.3 eV is
carbon sp3 hybrid C–C/C–H bond.36 High resolution spectrum
Fig. 2 Effects of pH value on the fluorescence intensity of a BQDs
of N 1s (Fig. S3b, ESI†) shows that the signal peaks at 400.03 eV solution.
and 399.38 eV are respectively the signal peaks of pyrrole N and
pyrimidine N.37 It can be seen from the high-resolution spec-
trum of O 1s (Fig. S3c, ESI†) that the main bonding of O is O–H
(533.3 eV), O–N (532.3 eV), C]O (531.5 eV) and C–O (530.8 eV).38
Above results show that the surface of BQDs is rich in hydro-
philic groups and has good water solubility, which is consistent
with the infrared spectrum results.
anti-salt properties, and can be used as uorescent probes to
detect bioactive molecules in vivo and in vitro. The effect of
solution pH value on the uorescence intensity of BQDs was
also investigated. As shown in Fig. 2, when the pH value is
within the range of 2.0–3.0, the uorescence intensity of BQDs
solution increases with the increase of the pH value. When pH
value is within the range of 10.0–13.0, uorescence intensity
decreases gradually with the increase of pH value of solution.
When pH is within the range of 3.0–10.0, uorescence intensity
basically remains unchanged with the increase of pH value of
the solution.
3.2 Optical performance of the BQDs
To explore the optical properties of BQDs, UV-vis absorption
and uorescence spectra of BQDs were studied. The blue curve
in Fig. 1d is the UV-vis absorption spectrum of BQDs, which has
the maximum absorption peak at 280 nm. The black curve in
Fig. 1d is the uorescence excitation spectrum of BQDs, with
the maximum excitation wavelength of 446 nm. Under the
excitation of this wavelength light, the maximum emission peak
of BQDs appears at 518 nm (red curve). In addition, the uo-
rescence emission of BQDs at different excitation wavelengths
was investigated. The results are shown in Fig. S4 (ESI).† The
uorescence emission maximum of BQDs was red shied with
the increase of excitation wavelength, indicating that the uo-
rescence emission spectrum of the BQDs was excitation
dependent. At the maximum excitation wavelength of 446 nm,
the maximum emission wavelength was 518 nm.
3.4 Characterization of DMI
DMI was synthesized by a two-step nucleophilic reaction. The
structure of DMI was characterized by mass spectrometry (MS),
and the result is shown in Fig. S7 (ESI).† It can be seen from the
gure that 305.07 m/z is the cationic MS peak from DMI, and the
abundance of 305.7 m/z ion is very high, indicating that the
purity of the synthesized DMI is very high. In addition, the
Using quinine sulfate as the reference, the QY of prepared
BQDs was obtained to be 14.9%, which is much higher than that
of many reported BQDs that were prepared using coffe grounds
(3.8%),39 watermelon peel (6.7%)40 and sweet potatoes (2.8%)41
as precursor.
3.3 Stability of BQDs solution
In order to evaluate the stability of BQDs solution, the effects of
UV lamp exposure time and solution ion strength on the
stability of BQDs were investigated. The uorescence intensity
of BQDs solution was basically unchanged (Fig. S5, ESI†) aer
exposure to UV lamp (365 nm) for 2 h, indicating that the BQDs
solution had good light resistance and bleaching resistance.
Fig. S6 (ESI†) shows the effect of ionic strength of the solution
on the uorescence intensity of BQDs. It was found that the
uorescence intensity of BQDs solution was basically
unchanged in 0–2.5 M sodium chloride solution. The above
Fig. 3 (a) The fluorescence emission spectrum of BQDs; (b) the UV-
visible absorption spectrum of BQDs; (c) the overlap of fluorescence
emission spectrum of BQDs and the absorption spectrum of DMI; and
(d) fluorescence spectra of BQDs in the presence of DMI at different
results indicate that the BQDs have good anti-bleaching and concentrations.
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compound was further identied by 1H NMR and 13C NMR, and
the results are shown in Fig. S8 and S9 (ESI),† conrming DMI
identity. The uorescence spectrum and UV-visible absorption
spectrum of DMI were studied. As shown in Fig. 3a, the
maximum emission wavelength of DMI is at 595 nm. Fig. 3b
shows the UV-visible absorption spectrum of DMI. It can be
seen from the gure that DMI has the maximum absorption
peak near 549 nm.
3.5 FRET between BQDs and DMI
FRET between BQDs and DMI was investigated. The results
show that the uorescence emission spectrum of BQDs and
the absorption spectrum of DMI overlap effectively (Fig. 3c),
which suggests that when the two coexist FRET will occur.
Moreover, with the increase of DMI concentration, the uo-
rescence intensity of BQDs gradually decreased, while that of
Fig. 5 Specificity of the proposed ratiometric fluorescence assay
ꢀ
based on FRET for HSO3 detection. Concentration ¼ 100 mM for all
species tested.
ꢀ
DMI gradually increased (Fig. 3d). When HSO3 is present in
the system, it reacts with DMI, which breaks up the conjugate
double bond in DMI (Fig. S10, ESI†), reducing the absorption
intensity of DMI (Fig. S11, ESI†) and thus resulting in
a decrease in FRET efficiency and recovery of uorescence
emission of BQDs (Fig. S12, ESI†). The results demonstrate
that this FRET system is caꢀpable of monitoring and quanti-
fying in endogenous HSO3 through a ratiometric uores-
cence response.
3.7 Selectivity investigation
In order to assess the selectivity of the proposed assay, the
effects of various anions, reactive oxygen species, reducinꢀg
substances and biological mercaptan on the detection of HSO3
were investigated. The results are shown in Fig. 5. When Clꢀ,
Brꢀ, Iꢀ, NO2ꢀ, NO3ꢀ, HPO42ꢀ, CH3COOꢀ, CO32ꢀ, HCO3ꢀ, H2O2,
GSH, Cys, S2ꢀ and ascorbic acid (AA) were introduced into the
system, the ratio uorescence intensity of the system did not
change signicantly as compared with blank signal. When
HSO3ꢀ was added to the system at the same concentration, the
ratio uorescence intensity of the system increased signi-
ꢀ
3.6 Ratiometric uorescence detection of HSO3
To evaluate the FRET system for ratiometric uorescence assay
ꢀ
of HSO3ꢀ, the response time of HSO3 to the system and the
cantly. These results indicate that the assay has a very good
linear range of the response were investigated. The results are
shown in Fig. S13 (ESI†) and 4. As can be seen from the gure,
aer adding HSO3ꢀ the ratio uorescence intensity (F518/F595) of
the system increases gradually with the extension of reaction
time, and reaches a maximum aer 10 min. With the increase of
HSO3ꢀ concentration, uorescence intensity of BQDs at 518 nm
increases and uorescence intensity of DMI at 595 nm
decreases gradually. F595/F518 value and HSO3ꢀ concentration in
the range of 1.0–225 mM show a good linear relationship with
a linear regression equation: F518/F595 ¼ 0.00919C + 0.4379
ꢀ
selectivity for HSO3
.
ꢀ
3.8 The detection of HSO3 in cells lysates
To demonstrate thꢀe feasibility of the proposed method for the
detection of HSO3 in real biological samples, we performed
HSO3ꢀ assay with lysates of HepG2, 7702 and MCF-ꢀ7 cells. Each
sample was measured ve times in parallel. HSO3 concentra-
tion in HepG2, 7702 and McF-7 cell lysates (1.43 ꢂ 106 cells per
mL) was found 36.8, 34.2 and 30.8 mM, respectively. RSDs of the
ve parallel assays for HepG2, 7702 and McF-7 cell lysates were
ꢀ
(where C is concentration of HSO3 in mM), R2 ¼ 0.9919. The
detection limit was estimated to be 0.5 mM (S/N ¼ 3).
Table 1 Analytical results of HSO3ꢀ in cells lysates (diluted 10 times)
Sample
(cell)
Found
(mM)
Added
(mM)
Found total
(mM)
Recovery
(%)
HepG2
3.68
5.0
10.0
20.0
5.0
10.0
20.0
5.0
8.65
13.37
23.59
8.51
13.02
23.24
8.27
99.4
96.9
99.6
101.8
96.0
99.1
103.8
103.4
95.7
7702
3.42
3.08
ꢀ
Fig.
4
Ratiometric fluorescence assay of HSO3
:
fluorescence
ꢀ
spectra of BQDs-DIM FRET system in the presence of HSO3 at
different concentrations (a), and a calibration curve for HSO3 quan-
MCF-7
10.0
20.0
13.42
23.22
ꢀ
tification (b).
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0.3%, 0.6% and 0.9%, respectively. The spike and recovery tests 10 R. F. McFeeters and A. O. Barish, J. Agric. Food Chem., 2003,
were carried out in cell lysates. The recovery was between 95– 51, 1513–1517.
104% (Table 1), indicating that this method was well suited for 11 G. Jankovskiene, Z. Daunoravicius and A. Padarauskas, J.
ꢀ
the determination of HSO3 in cell lysates.
Chromatogr. A, 2001, 934, 67–73.
12 Y. Sun, P. Wang, J. Liu, J. Zhang and W. Guo, Analyst, 2012,
137, 3430–3433.
13 H. Xie, F. Zeng, C. Yu and S. Wu, Polym. Chem., 2013, 4,
5416–5424.
4. Conclusions
A ratiometric uorescence assay based on RET between BQDs
and organic dye (DMI) was developed for the detection of sulfur
dioxide derivatives. The proposed method was applied to the
quantication of HSO3ꢀ in HepG2, 7702 and McF-7 cell lysates.
The assay has several signicant advantages. First, this assay is
based on the principle of FRET with BQDs as the energy donor.
It's well known that there is no photobleaching when BQDs is
irradiated by the excitation light, which makes the FRET system
very stable. Secondly, using ratio uorescence analysis to detect
the target eliminates the impacts of environmental changes and
of the instability of the test instrument. Thirdly, this assay has
a good selectivity. Many co-existing anions, reactive oxygen
species, reducing substances and biological mercaptan in the
biological samples have no interference with the detection of
HSO3ꢀ. Considering these advantageous characteristics, we
anticipate the proposed ratiometric uorescence assay will be
very useful for physiological and pathological studies of SO2.
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Acknowledgements
Financial support from the Natural Science Foundation of
China (No. 201575031 to SZ), the U.S. National Institutes of
Health (GM089557 to YML) and Natural Science Foundation of
Guangxi Province (No. 2017GXNSFFA198014) as well as BAGUI
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