A Self-Assembled Ratiometric Polymeric Nanoprobe for Highly Selective Fluorescence Detection of Hydrogen Peroxide

Article abstract of DOI:10.1021/acs.langmuir.7b00189

In this study, a dual-emission fluorescence resonance energy transfer (FRET) polymeric nanoprobe by single-wavelength excitation was developed for sensitive and selective hydrogen peroxide (H₂O₂) detection. Polymeric nanoprobe was prepared by simple self-assembly of functional lipopolymers, which were 4-carboxy-3-fluorophenylboronic acid (FPBA)-modified DSPE-PEG (DSPE-PEG-FPBA) and 7-hydroxycoumarin (HC)-conjugated DSPE-PEG (DSPE-PEG-HC). Subsequent binding of alizarin red S (ARS) to FPBA endowed the nanoprobe with a new fluorescence emission peak at around 600 nm. Because of the perfect match of the fluorescence emission spectra of HC with the absorbance spectra of ARS-FPBA, FRET was achieved between them. The sensing strategy for H₂O₂ was based on H₂O₂-induced deboronation reaction and boronic acid-mediated ARS fluorescence. Interaction between phenylboronic acid and ARS was revisited herein and it was found that electron-donating or -withdrawing group on phenylboronic acid (PBA) has significant influence on the fluorescence property of ARS, which enabled sensitive and selective H₂O₂ sensing. The nanoprobe displayed two well-separated emission bands (150 nm), providing high specificity and sensitivity for ratiometric detection of H₂O₂. Further application was exploited for the determination of glucose and the results demonstrated that the proposed strategy showed ratiometric response capability for glucose detection. The current method does not involve complicated organic synthesis and opens a new avenue for the construction of multifunctional polymeric fluorescent nanoprobe.

Full text of DOI:10.1021/acs.langmuir.7b00189

Article
pubs.acs.org/Langmuir
A Self-Assembled Ratiometric Polymeric Nanoprobe for Highly
Selective Fluorescence Detection of Hydrogen Peroxide
Chongchong Feng, Fengyang Wang, Yijing Dang, Zhiai Xu,* Haijun Yu, and Wen Zhang
†
†
†
,†
‡
†
†
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, People’s Republic of China
Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic
‡
of China
*
S Supporting Information
ABSTRACT: In this study, a dual-emission fluorescence resonance energy transfer (FRET) polymeric nanoprobe by single-
wavelength excitation was developed for sensitive and selective hydrogen peroxide (H O ) detection. Polymeric nanoprobe was
2
2
prepared by simple self-assembly of functional lipopolymers, which were 4-carboxy-3-fluorophenylboronic acid (FPBA)-modified
DSPE-PEG (DSPE-PEG-FPBA) and 7-hydroxycoumarin (HC)-conjugated DSPE-PEG (DSPE-PEG-HC). Subsequent binding
of alizarin red S (ARS) to FPBA endowed the nanoprobe with a new fluorescence emission peak at around 600 nm. Because of
the perfect match of the fluorescence emission spectra of HC with the absorbance spectra of ARS-FPBA, FRET was achieved
between them. The sensing strategy for H O was based on H O -induced deboronation reaction and boronic acid-mediated
2
2
2
2
ARS fluorescence. Interaction between phenylboronic acid and ARS was revisited herein and it was found that electron-donating
or -withdrawing group on phenylboronic acid (PBA) has significant influence on the fluorescence property of ARS, which
enabled sensitive and selective H O sensing. The nanoprobe displayed two well-separated emission bands (150 nm), providing
2
2
high specificity and sensitivity for ratiometric detection of H O . Further application was exploited for the determination of
2
2
glucose and the results demonstrated that the proposed strategy showed ratiometric response capability for glucose detection.
The current method does not involve complicated organic synthesis and opens a new avenue for the construction of
multifunctional polymeric fluorescent nanoprobe.
17−19
INTRODUCTION
ment.
In particular, ratiometric probes with large emission
■
shift upon single-wavelength excitation are highly desired.
In past years, nanomaterial-based ratiometric fluorescent
probes have drawn increasing interests owing to their
remarkable advantages including their nanosize, good photo-
Hydrogen peroxide (H O ) is a crucial biochemical molecule,
2
2
1
−4
which plays a significant role in cellular signal transduction.
In the presence of O and enzymes, lots of biological substrates
can be specifically catalyzed to produce H O as a byproduct.
However, excessive presence of H O is associated with
oxidative stress and serious cell damage, leading to pathogenic
states such as inflammation, cancer, cardiovascular diseases, and
neurodegenerative disorders.
have been devoted for selective and sensitive detection of
2
5
2
2
stability, and rich functional groups for further modifica-
20−22
2
2
tion.
The polymeric micelle is one promising nanosystem
receiving much attention in many fields especially that of drug
23−25
delivery as carriers of diagnostic and therapeutic agents.
6
,7
Therefore, extensive efforts
Polymeric micelles are usually core−shell structures formed by
self-assembly of amphiphilic block copolymers present with
attractive features, such as good solubility and prolonged blood
8
−11
H O .
Among the various modalities, fluorescence
2
2
26−30
detection is a fascinating method characterized by fast response,
high sensitivity, and compatible with microscopic techni-
que.
developed in recent years, the majority of them offer single-
emission fluorescent signal, which may lead to inaccurate
quantification. Ratiometric fluorescent probes that can exhibit
strong anti-interference ability and afford high contrast images
are favorable for H O detection in biological environ-
circulation.
In addition, they offer the benefit of high
versatility in terms of composition and functionality, providing
high potential for designing nanoprobes for bimolecular
detection.
1
2−16
Although some fluorescent probes have been
Received: January 18, 2017
Revised: March 20, 2017
2
2
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Scheme 1. Schematic Illustration of the Self-Assembled FRET Polymeric Nanoprobe Based on ARS-FPBA for Ratiometric
Detection of H O2
2
Alizarin red S (ARS) is a readily available dye which is nearly
EXPERIMENTAL SECTION
■
nonfluorescent due to the excited state proton transfer
Chemicals and Reagents. FPBA, ARS, 2,2-azobis(2-methylpro-
pionamidine) dihydrochloride (AAPH), potassium dioxide (KO ), and
boric acid (BA) were all purchased from Aladdin reagent company
Shanghai, China). Glucose oxidase (GOx), 4-carboxyphenylboronic
acid (4-CPBA, 98%), 4-mercaptophenylboronic acid (TPBA, 90%), 3-
2-hydroxy-1-methylethyl-2-nitrosohydrazino)-1-propanamine
NOC5), fructose, galactose, sucrose, maltose, anhydrous glucose, N-
3
1,32
quenching.
Its fluorescence intensity increases dramatically
2
33
upon forming adducts with boronic acid derivatives. This
excellent property was adopted for many fluorescent sensing
applications including mainly carbohydrates based on their
(
(
(
34−36
competitive binding to boronic acid derivatives.
However,
the applications are limited and they usually give turn-off
fluorescence responses. In this study, by screening boronic acid
derivatives with the absorbance and fluorescence spectra we
found that the fluorescence response to H O was dependent
acetylneuraminic acid (SA), and fetal bovine serum (FBS) were all
purchased from Sigma-Aldrich (Shanghai, China). PBA (99%), 3-
aminophenylboronic acid (APBA, 98%), 3-carboxyphenylboronic acid
(3-CPBA, 98%), 1-hydroxybenzotriazole anhydrous (HOBT, 98%),
2
2
and N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochlor-
ide (EDCI, 99%) were all purchased from J&K Scientific Ltd.
on the functional group on phenylboronic acid (PBA) and the
response was sensitive and highly selective in the case of 4-
carboxy-3-fluorophenylboronic acid (FPBA). To the best of our
knowledge, there is little to no information in the literature
about utilizing ARS-boronate composite for H O sensing.
(Shanghai, China). 7-Hydroxycoumarin-3-carboxylic acid (HC, >98%)
was purchased from TCI Co., Ltd. (Shanghai, China). 1,2-Distearoyl-
sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-
2
000] (DSPE-PEG-NH ) was purchased from Shanghai A. V. T.
2
2
2
Pharmaceutical L.T.D. Co. (Shanghai, China). HEPES buffer (25 mM,
pH 7.4) and Milli-Q water were used throughout the whole study.
Dialysis tubes with a molecular weight cutoff of 1000 Da (MWCO
Moreover, it does not involve complicated synthesis and
purification processes. On the basis of the self-assembly
method, we developed a fluorescence resonance energy transfer
1
000 Da) were obtained from Spectrum Laboratories, Inc. (CA,
(
FRET) polymeric nanoprobe with dual-emission for highly
U.S.A.).
Measurements. The UV−vis absorption and fluorescence spectra
selective detection of H O by modifying FPBA and 7-
hydroxycoumarin-3-carboxylic acid (HC) on lipopolymer 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino-
(
2
2
were measured with a Cary 60 UV−vis spectrophotometer and a Cary
Eclipse spectrofluorophotometer, respectively (Agilent Technologies,
1
CA, U.S.A.). H NMR of DSPE-PEG-HC and DSPE-PEG-FPBA were
polyethylene glycol)-2000] (DSPE-PEG-NH ). The nanop-
2
measured by using a Bruker-400 MHz NMR spectrometer at 25 °C.
The dynamic light scattering (DLS) measurement was measured at 25
°C using a Malvern zetasizer Nano ZS (ZS90, Malvern, U.K.).
Transmission electron microscope (TEM) image was obtained with a
JEOL JEM-2100 microscopy (JEOL, Tokyo, Japan). The mass
spectrometry was conducted by a Micromass Q-TOF Ultima mass
spectrometer (Waters, Manchester, U.K.). Fluorescence lifetime
measurements were performed on a FLS980 fluorescence spectrom-
eter from Edinburgh Instruments Ltd. (Photonics Division, U.K.).
robe was successfully used to determine H O ratiometrically
2
2
with two well-separated emissions upon single-wavelength
excitation, and the fluorescence detection of glucose based on
enzyme-catalyzed H O production was realized. This
developed method was facile, highly selective, and promising
as a candidate for detection of H O and H O -related
2
2
2
2
2
2
substances.
B
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 1. Absorbance (A) and fluorescence (B) spectra of ARS in the presence of different concentrations of PBA (λ = 488 nm). (C) Ratiometric
ex
change of ARS absorbance (A /A ) in the presence of BA or PBA derivatives. (D) Fluorescence intensity change at 600 nm as a function of BA or
2
1
PBA derivatives concentration in the range of 0−1.2 mM. (F , the fluorescence intensity of ARS; F, the fluorescence intensity of ARS-BA and ARS-
0
PBA derivatives; [ARS] = 0.1052 mM.).
Synthesis of DSPE-PEG-FPBA and DSPE-PEG-HC. First, FPBA
was dissolved in DMSO and activated by EDCI and HOBT for 2 h
solution (0.6 mM). The impurities were then separated from the
solution by dialysis (MWCO: 1000 Da) for 2 h.
(
FPBA/EDCI/HOBT = 1:1.2:1.2, mol/mol/mol). Second, DSPE-
The Stability of the Ratiometric Nanoprobe. The stability of
the nanoprobe was assessed by monitoring the change of micellar size
under simulative physiological conditions. In brief, the micellar
solution was mixed with an equal volume of HEPES buffer (25 mM,
pH 7.4) and medium containing 10% fetal bovine serum (FBS). The
mixtures were incubated at 37 °C for different times and determined
by DLS.
PEG-NH was dissolved in DMSO, mixed with TEA (DSPE-PEG-
NH /TEA/FPBA = 1:3:3, mol/mol/mol) and stirred for 2 h. Then,
FPBA solution was dropwise added to the solution of DSPE-PEG-
NH , reacted at room temperature for 24 h. The mixture was dialyzed
for 72 h to remove unreacted impurities and then lyophilized. The
chemical structure of DSPE-PEG-FPBA was confirmed by H NMR
spectra. H NMR (500 MHz, DMSO-d , δ): 7.63 (s, 1H), 7.57 (s,
1
2
2
2
1
1
Glucose Detection. Glucose detection was performed as follows:
6
6
μL of 2 mg/mL GOx, 6 μL of different concentrations of glucose
H), 6.53 (s, 1H), 5.64 (s, 1H), 5.10−5.06 (m, 1H), 4.28 (s, 1H), 3.86
ο
and 18 μL of HEPES buffer (25 mM, pH 7.4) were incubated at 37 C
for 30 min. Then 30 μL of the nanoprobe solution was injected into
the resulting solution and allowed to incubate further for 1 h.
Subsequently, the fluorescence signal of the mixtures was recorded.
The detection of glucose in fetal bovine serum was carried out as
follows: the fetal bovine serum was first treated with 10 mg/mL GOx
(
3
s, 6H),3.80−3.67 (m, 5H), 3.65 (s, 8H), 3.62−3.48 (m, 72H), 3.44−
.34 (m,6H), 3.32 (s, 20H), 3.09−3.05 (m, 1H), 2.64 (s, 12H), 2.54−
.47 (m,73H), 2.37 (s, 2H), 2.28−1.45 (m, 25H), 1.50 (s, 13H), 1.50
2
(
(
s, 18H), 1.24 (s, 22H), 1.15 (s, 1H), 0.86 (t, J = 6.7 Hz, 23H), 0.01
s, 3H).
DSPE-PEG-HC was synthesized by following a similar procedure as
ο
for 12 h at 37 C. Then, glucose with varying concentrations was
that of DSPE-PEG-FPBA. The chemical structure of DSPE-PEG-HC
was confirmed by H NMR spectra. H NMR (500 MHz, DMSO-d ,
δ): 9.17 (s, 1H), 8.79−8.75 (m, 1H), 8.30 (s, 1H), 8.21 (s,1H), 8.17−
spiked into the pretreated fetal bovine serum. Then, the glucose
detection was carried out according to the procedure as mentioned
above.
1
1
6
8
1
1
2
5
3
2
1
.13 (m, 1H), 8.08−8.04 (m, 1H), 7.91 (s, 1H), 7.48 (d, J = 8.3 Hz,
H), 7.38 (dd, J = 21.5, 14.0 Hz, 2H), 7.22 (s, 1H), 7.16 (d, J = 9.0 Hz,
H), 6.97 (s, 1H), 6.92 (s, 1H), 6.06 (s, 1H), 5.74 (s, 1H), 4.30 (s,
0H), 4.28 (s, 24H), 4.18 (d, J = 99.1 Hz, 29H), 3.75 (d, J = 93.8 Hz,
H), 3.86−3.48 (m, 29H), 3.42 (s, 1H), 3.31 (d, J = 11.6 Hz, 13H),
.22 (s, 1H), 2.89 (s, 1H), 2.64 (s, 3H), 2.51 (s, 62H), 2.37 (s, 1H),
.26 (s, 1H), 2.11−2.07 (m, 1H), 1.51−1.47 (m, 1H), 1.24 (s, 8H),
.15 (s, 3H), 0.86 (t, J = 6.8 Hz, 2H), 0.01 (s, 3H).
RESULTS AND DISCUSSION
■
Polymeric micelles are unique nanostructures that can integrate
multifunctionalities such as recognition, detection, and drug
delivery. DSPE-PEG is a biocompatible amphiphilic polymer.
Herein, we utilize DSPE-PEG to construct a ratiometric
fluorescent nanoprobe for H O2 detection by conjugating
2
Preparation of the Ratiometric Nanoprobe. The ratiometric
FPBA and HC on DSPE-PEG polymer (Scheme 1). The
nanoprobe was prepared through a film hydration process. Briefly, 9
mg of DSPE-PEG, 1 mg of DSPE-PEG-FPBA, and 0.005 mg of DSPE-
PEG-HC were mixed in 5 mL of chloroform. Then the solution was
evaporated to remove chloroform. Five milliliters of HEPES buffer (25
mM, pH 7.4) was added to the round-bottom flask and sonicated for
detection strategy is based on H O -induced deboronation
2
2
reaction and boronic acid-mediated ARS fluorescence. As
illustrated in Scheme 1, DSPE-PEG-FPBA, DSPE-PEG-HC,
and DSPE-PEG can self-assemble to form the polymeric
nanoprobe. In the absence of ARS, the polymeric nanoprobe
3
0 min. One milliliter of micelle solution was mixed with 1 mL of ARS
C
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
displays a blue-colored fluorescence with an emission peak at
F
F
{1 + kK[B]}
{1 + K[B]}
=
450 nm from HC upon excitation at 405 nm. After ARS is
(1)
0
added into the micelle solution, it interacts with FPBA and
FRET could occur between ARS-FPBA and HC with the
appearance of a new fluorescence emission peak at 600 nm.
However, in the presence of H O , ARS would dissociate from
where F and F are the fluorescence intensities of ARS in the
presence and absence of BA or PBA derivatives, respectively,
and k (= k /k ) represents the ratio of proportionality constants
connecting the fluorescence intensity and concentrations of the
species (complex, k ; free ligand, k ). K is the binding constant
for complexation of ARS with BA or PBA derivatives. The free
BA or PBA derivatives concentration, [B], can be related to
known total concentrations of BA or PBA derivatives
concentration (B ) and ligand (L ), by the following equation
0
c
L
2
2
the nanoprobe surface and result in the decrease of fluorescence
intensity at 600 nm and increase of fluorescence intensity at
c
L
450 nm, allowing a ratiometric fluorescence response for the
detection of H O by single-wavelength excitation.
2
2
It is known that ARS can form adducts with boronic/boric
0
0
acid and ARS shows great fluorescence enhancement upon
3
7
binding. Herein, we revisit this reaction by examining the
absorption and fluorescence spectra of ARS in the presence of
different boronic acid derivatives. As an example, Figure 1A
shows the absorption spectra of ARS in the absence and
presence of various concentrations of PBA. ARS displays two
obvious absorbance bands with peaks located at 333 and 510
nm in the range of 300−700 nm. Upon the addition of PBA,
the absorption at 333 nm declines significantly with a slight
blue-shift. The absorption peak at 510 nm blue shifts to 460 nm
gradually and the absorbance intensity decreases first but
increases later. Two isosbestic points at 386 and 488 nm
appear, indicating ARS and PBA have formed a stable
compound. Figure 1B shows the corresponding fluorescence
spectra of ARS without or with different concentrations of PBA.
This result clearly reflects that ARS has very weak fluorescence
emission but upon interaction with PBA, its fluorescence
increases rapidly with the maximum emission wavelength blue
shifting from 600 to 590 nm. To better understand the
interaction of ARS with boronic acid derivatives, we studied six
PBA derivatives together with BA by measuring the absorbance
{L K[B]}
0
B0 = [B] + {
1 + K[B]}
(2)
Together, eqs 1 and 2 describe the system. The data in
Figure 1D can be fitted well with the equations and Table 1
shows the results of the binding constant of ARS with BA or
PBA derivatives. The sequence of binding constant is FPBA >
PBA > 4-CPBA > APBA > 3-CPBA> TPBA > BA.
Table 1. Binding Constant of ARS with BA or PBA
Derivatives
4
-
3-
PBA
2624
APBA CPBA CPBA TPBA FPBA
BA
binding
1713 1860 1121 715 2804
213
constant
−1
(
M
)
According to previous studies, boronic groups in boronate-
derivatized organic compounds can be transformed into phenol
upon the reaction with H O .
4
0,41
We deduce that ARS-PBA
2
2
and fluorescence spectra. As shown in Figure 1C, A is the
derivatives can be used to detect H O . The spectroscopic
1
2
2
declined intensity of absorbance peak at about 510 nm and A2
is the increased intensity of absorbance peak at about 460 nm.
By increasing the concentration of BA or PBA derivatives, A2/
A1 rises rapidly and tends to approach constant at higher
concentration. In particular, the A /A ratio of ARS-APBA
approaches constant quickly while ARS-TPBA keeps gradual
increase all through the investigated concentration range.
Accordingly, a significant color change from pink to orange was
observed in the presence of PBA derivatives (Figure S1,
Supporting Information). The change in fluorescence is plotted
against BA or PBA derivatives concentration (Figure 1D).
Upon the addition of different concentrations of BA or PBA
derivatives to the solution of ARS, BA and six PBA derivatives
behave similarly, whereas the fluorescence enhancement differs
greatly. For instance, there is about 150-fold fluorescence
properties of ARS-PBA derivatives after reaction with H O
2 2
were investigated in HEPES buffer (25 mM, pH 7.4). As shown
in Figure 2A,B, UV−vis spectra indicates that the absorption
peak of ARS-FPBA at 510 nm red shifts to 515 nm with slight
enhancement along with the increase of H
The fluorescence spectra shows that the fluorescence intensity
of ARS-FPBA decreases with the H O concentration increase
O concentration.
2
1
2
2
2
2
ranging from 0 to 500 μM. The fluorescence change of ARS-
FPBA (F/F ) at 600 nm is plotted in Figure 2C and it shows
0
that F/F0 quickly decreases and levels off when the
concentration of H O is above 500 μM. The reaction of
2
2
ARS-FPBA with H O transformed boronates into phenols
2
2
with concomitant dissociation of BA and ARS (Scheme S1,
Supporting Information). FPBA shows much higher enhance-
ment on ARS fluorescence than BA, therefore, H O -induced
2
2
deboronation reaction leads to decreased fluorescence
response. To support the above-mentioned mechanism, the
solution of ARS-FPBA before and after reaction with H O
increase (F/F ) for FPBA but only a 10-fold increase is
0
observed for that of APBA. We propose that the disparity of the
enhancement on ARS fluorescence originates from the
difference of the substitution groups on phenyl ring. Carboxyl
and fluorine are electron-withdrawing groups that are better at
preventing the excited state proton transfer quenching of ARS
than electron-donating groups, such as amino and thiol groups.
2
2
were detected by mass spectrometry (MS). Upon reaction with
H O , a new peak ascribed to 4-carboxy-3-fluorophenol
2
2
appeared (Figure S2, Supporting Information).
As expected, the fluorescence intensity of ARS-CPBA and
ARS-FPBA both decreases upon reaction with H O . In
2
2
Additionally, the difference of F/F between ARS-3-CPBA and
0
obvious contrast, the fluorescence intensity of ARS-BA is
ARS-4-CPBA indicates that para-substituent is better for
fluorescence enhancement.
almost unchanged in the presence of H O but that of ARS-
2
2
APBA is enhanced. Similarly, after reacting with H O , F/F of
2
2
0
The binding affinity of ARS with BA and PBA derivatives was
analyzed by nonlinear least-squares regression with the
following equations
ARS-TPBA has a trend of rise, while the subsequent decline
may be explained by the susceptibility of thiol group to be
oxidized (Figure 2D). Therefore, the variation in fluorescence
3
8,39
D
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 2. (A) Absorbance and (B) fluorescence spectra of ARS-FPBA in the presence of different concentrations of H O . (C) Fluorescence
2
2
intensity change of ARS-FPBA at 600 nm as a function of H O concentration. ([ARS] = 0.0526 mM, [FPBA] = 0.0526 mM). (D) The fluorescence
2
2
intensity change of ARS-BA or ARS-PBA derivatives with the addition of different concentrations of H O (F , the fluorescence intensity without
2
2.
0
addition of H O ; F, the fluorescence intensity with addition of H O ).
2
2
2
2
Figure 3. (A) The TEM image of the micelle nanoparticles. The scale bar corresponds to 100 nm. (B) Absorption spectra of ARS-FPBA and
emission spectra of HC. (C) The fluorescence spectra of different polymeric nanoprobes. (λex = 405 nm). (D) Fluorescence photostability of the
polymeric nanoprobe in HEPES buffer solution (25 mM, pH 7.4).
E
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 4. Stability of the polymeric nanoprobe in (A) HEPES buffer solution (25 mM, pH 7.4) and (B) HEPES buffer solution (25 mM, pH 7.4)
containing 10% FBS studied by dynamic light scattering experiment.
intensity for different PBA derivatives allows us to rationally
design H O -responsive fluorescent probe, and FPBA was
chosen for the construction of the nanoprobe.
emission spectra of the polymeric nanoprobe in the presence of
ARS by varying ARS concentrations (Figure S5, Supporting
Information). There was continuous decrease in fluorescence
intensity assigned to HC, meanwhile, the fluorescence intensity
of ARS-FPBA increased gradually and tended to be constant
with the increase of ARS concentration, indicating that ARS
was excessive relative to the polymeric nanoprobe. All the
results suggested that FRET-based nanoprobe was successfully
prepared. The photostability of the polymeric nanoprobe was
tested and the results are shown in Figure 3D. When excited at
405 nm, the emission intensity stays almost unchanged over
time, suggesting the good photostability of the polymeric
nanoprobe.
2
2
HC and FPBA was conjugated with DSPE-PEG-NH2
through condensation reaction between amino and carboxyl
group (Scheme 1 and S2, Supporting Information). The
1
successful synthesis of them was verified by H NMR spectra
(Figure S3, Supporting Information). Next, we prepared the
polymeric nanoprobe by hybrid self-assembly of DSPE-PEG,
DSPE-PEG-HC and DSPE-PEG-FPBA through a film hydra-
tion process. Transmission electron microscope (TEM) image
shows that the micelle nanoparticles are of spherical-like
structure with diameter of about 40 nm (Figure 3A). HC was
chosen for construction of FRET nanoprobe with ARS-FPBA
because the absorption peak of ARS-FPBA is at 455 nm,
perfectly overlapping with the emission spectra of HC (450
nm) (Figure 3B). In order to prove the occurrence of FRET,
we used DSPE-PEG with DSPE-PEG-FPBA, DSPE-PEG-HC,
or both DSPE-PEG-HC and DSPE-PEG-FPBA to construct
three kinds of polymeric nanoprobes. As shown in Figure 3C,
the fluorescence intensity of the ratiometric nanoprobe at 450
nm attributed to HC decreases, while the fluorescence intensity
at 600 nm ascribed to ARS-FPBA increases. We obtained a
series of FPBA/HC nanoparticles with different ratios of FPBA
and HC by changing the amounts of DSPE-PEG-FPBA and
DSPE-PEG-HC. Along with increase of the ratio of FPBA to
HC, the fluorescence intensity of ARS would increase while
that of HC would decrease (Figure S4, Supporting
Information). In the presence of H O , the fluorescence
The decay profiles of the polymeric nanoprobe before and
after conjugation to ARS are shown in Supporting Information
Figure S6. The average lifetimes of the polymeric nanoprobe
before and after conjugation to ARS are 4.75 and 3.99 ns,
42
respectively. According to eq 3, where τda and τ are the
d
fluorescence lifetimes of a donor in the presence and absence of
an acceptor, respectively, the FRET efficiency was estimated to
be 0.16 for the polymeric nanoprobe
τ
τ
da
E = 1 −
(3)
d
Good stability is crucial for the application of the polymeric
nanoprobe. We thus evaluated the stability of the polymeric
nanoprobe in both HEPES buffer solution and serum-
containing HEPES. The polymeric nanoprobe shows good
dispersity in either serum-free or serum-containing HEPES for
24 h, implying good dispersion stability of the polymeric
nanoprobe as confirmed by dynamic light scattering measure-
ment (Figure 4). Using fluorescein as a standard, the
fluorescence quantum yield (QY) of the polymeric nanoprobe
was calculated to be around 2.4%.
2
2
intensity of HC was expected to increase while that of ARS
would decrease. In order to magnify the ratiometric change of
these two peaks, we chose 200:1 as the optimum concentration
ratio and the dual-emission fluorescence intensity ratio (F450
/
F600) equal to 1.5 approximately. Besides, in order to optimize
the concentration of ARS, we measured the fluorescence
F
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 5. (A) Ratiometric fluorescence response of the polymeric nanoprobe in the presence of different concentrations of H O . (B) The plot of
2
2
F450/F600 as a function of H O concentration. (C) Linear plots of F450/F600 at low concentrations of H O . (D) Selectivity investigation of the
2
2
2
2
polymeric nanoprobe in the presence of different ROS and RNS.
The fluorescence characteristics of the prepared polymeric
nanoprobe were then investigated. As expected, the polymeric
nanoprobe shows two well-separated fluorescence peaks at 450
and 600 nm upon excitation at 405 nm, which provides the
basis for ratiometric fluorescence detection of H O . Increasing
We compared our probe with the reported boronate probes
in recent years (Table 2). Our probe provides a wide detection
Table 2. Boronate Probes for the Detection of H O
2
2
2
2
detection range
product λem
(nm)
LOD
(μM)
the nanoprobe concentration, the fluorescence intensity ratio
F450/F600) decreased and tended to approach constant at
probe
(μM)
ref
(
dLys-AgNCs
T-CuNPs
NAC-AuNCs
probe 1
0.8−200
0.55−110
0.04−6.66
0−180
10−200
0−500
450/640
615
0.20
0.55
0.027
2.0
5
higher concentrations (Figure S7, Supporting Information).
8
Upon the addition of H O , the fluorescence intensity of peak
2
2
650
10
19
40
at 450 nm was attributed to HC increases, while the
fluorescence peak at 600 nm ascribed to ARS-FPBA
continuously decreases, resulting in the ratiometric response
410/542
500
TPE-BO
0.52
0.76
our work
450/600
to H O2 (Figure 5A). Figure 5B shows the plot of the
2
fluorescence intensity ratio (F450/F600) as a function of H O2
2
concentration. The ratio of F450/F600 gradually increases along
range and a comparable detection limit. Meanwhile, the
developed probe shows the advantages of facile preparation,
high selectivity, and good fluorescence properties.
with the increased H O concentration. The dual-emission
2
2
fluorescence intensity ratio increases linearly with the
concentration of H O between 0 and 500 μM (Figure 5C).
2
2
Oxidase-triggered oxidation of substrates could produce
H O2 as a byproduct. By taking advantage of H O as
The correlation coefficient is 0.9973 with a detection limit of
2
2
2
∼0.76 μM (based on a signal-to-noise ratio of 3). We examined
intermediate, the polymeric nanoprobe was configured into
biosensor for glucose. The fluorescence spectra of the
polymeric nanoprobe with various concentration of glucose in
the presence of GOx are depicted in Figure 6A. With increasing
amount of glucose, the emission of the polymeric nanoprobe at
600 nm is quenched gradually while that at 450 nm increases
dramatically. There is continual increase of F450/F600 with the
rising of glucose concentration, which indicates that the signal
change is related to the glucose concentration (Figure 6B). As
shown in Figure 6C, the relationship between F450/F600 and
the response of the polymeric nanoprobe to other reactive
oxygen species (ROS) and reactive nitrogen species (RNS) that
have interferences. As shown in Figure 5D, only the addition of
H O can obviously alter the fluorescence of the polymeric
2
2
•−
nanoprobe; other ROS and RNS, including superoxide (O ),
hypochlorite (ClO ), peroxynitrite (ONOO ), peroxyl radicals
ROO ), singlet oxygen ( O ), and nitric oxide (NO) display
2
−
−
•
1
(
2
small fluorescence response, suggesting high selectivity of the
polymeric nanoprobe for H O detection. In addition, we
2
2
2
investigated the response of the probe to H O under different
glucose concentration presents a good linear response (R =
2
2
pH (Supporting Information Figure S8), and the result
indicated that the probe showed an increased response to
H O along with increasing solution pH.
0.9863) in the range of 0.3−1 mM, with the detection limit of
10 μM (based on a signal-to-noise ratio of 3). Simultaneously,
the selectivity of the polymeric nanoprobe toward glucose was
2
2
G
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 6. (A) Ratiometric fluorescence response of the polymeric nanoprobe in the presence of different concentrations of glucose in the presence of
GOx. (B) The plot of F450/F600 as a function of glucose concentration. (C) Linear plots of F450/F600 at low concentrations of glucose. (D) Selectivity
investigation of the polymeric nanoprobe for the detection of glucose. The concentration of glucose is 1 mM, while those of other interferents are 0.5
mM.
investigated by detecting different carbohydrates and other
control substances (Figure 6D). No obvious responses are
observed for other related interferents including the high
concentration of glucose, which suggests the nanoprobe has
superior selectivity for glucose only in the presence of GOx.
The practical applicability of our study was tested by
detecting glucose in fetal bovine serum samples. The results are
shown in Table 3. It is observed that the recoveries range from
we believe that this proposed strategy may pave a new way for
the construction of a multifunctional polymeric fluorescent
nanoprobe and the nanoprobe holds great potential for
biochemical study.
ASSOCIATED CONTENT
■
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.lang-
muir.7b00189.
Table 3. Determination of Glucose in FBS Samples
sample
added (mM)
detected (mM)
recovery (%)
RSD (%)
Additional schemes, figures, and tables (PDF)
1
2
3
0.35
0.60
1.0
0.37
0.58
0.99
106
97
1.0
0.6
1.4
AUTHOR INFORMATION
■
99
ORCID
Zhiai Xu: 0000-0002-4391-2507
Notes
The authors declare no competing financial interest.
9
0
7% to 106%, and the relative standards deviations are from
.6% to 1.4%. These results indicate that this assay can be
further applied to the detection of glucose in real biological
samples.
ACKNOWLEDGMENTS
■
CONCLUSIONS
■
Financial supports by the National Natural Science Foundation
of China (No. 21675055, 31622025, 21375040, 21635003) are
greatly appreciated.
In summary, we developed a fluorescent nanoprobe by
modifying two fluorescent molecules of HC and ARS-FPBA
on lipopolymer and preparing nanoprobe by a simple self-
assembly method. The nanoprobe displayed selective and
ratiometric fluorescence response to H O . In combination
REFERENCES
■
2
2
(
1) Abo, M.; Urano, Y.; Hanaoka, K.; Terai, T.; Komatsu, T.;
with glucose oxidase, the nanoprobe was further applied to the
detection of glucose. The polymeric nanoprobe exhibited
remarkable features including facile preparation, long-term
stability, highly selective and ratiometric response. Therefore,
Nagano, T. Development of a highly sensitive fluorescence probe for
hydrogen peroxide. J. Am. Chem. Soc. 2011, 133, 10629−10637.
(2) Rhee, S. G. Cell signaling. H O , a necessary evil for cell signaling.
2
2
Science 2006, 312, 1882−1883.
H
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
(
3) Finkel, T. Signal transduction by reactive oxygen species. J. Cell
Biol. 2011, 194, 7−15.
4) Song, Z.; Kwok, R. T. K.; Ding, D.; Nie, H.; Lam, J. W. Y.; Liu,
(24) Talelli, M.; Rijcken, C. J. F.; van Nostrum, C. F.; Storm, G.;
Hennink, W. E. Micelles based on HPMA copolymers. Adv. Drug
Delivery Rev. 2010, 62, 231−239.
(
B.; Tang, B. An AIE-active fluorescence turn-on bioprobe mediated by
hydrogen-bonding interaction for highly sensitive detection of
hydrogen peroxide and glucose. Chem. Commun. 2016, 52, 10076−
(25) Duncan, R. The dawning era of polymer therapeutics. Nat. Rev.
Drug Discovery 2003, 2, 347−360.
(26) Nishiyama, N.; Kataoka, K. Current state, achievements, and
future prospects of polymeric micelles as nanocarriers for drug and
1
(
0079.
5) Liu, F.; Bing, T.; Shangguan, D.; Zhao, M.; Shao, N. Ratiometric
gene delivery. Pharmacol. Ther. 2006, 112, 630−648.
(27) Shi, Y.; van Nostrum, C. F.; Hennink, W. E. Interfacially
fluorescent biosensing of hydrogen peroxide and hydroxyl radical in
living cells with lysozyme−silver nanoclusters: lysozyme as stabilizing
ligand and fluorescence signal unit. Anal. Chem. 2016, 88, 10631−
hydrazone cross-linked thermosensitive polymeric micelles for acid-
triggered release of paclitaxel. ACS Biomater. Sci. Eng. 2015, 1, 393−
4
04.
1
(
0638.
6) Alfadda, A. A.; Sallam, R. M. Reactive oxygen species in health
and disease. J. Biomed. Biotechnol. 2012, 2012, 1−14.
7) Kamat, P. J.; Devasagayam, T. P. A. Oxidative damage to
(28) Kataoka, K.; Harada, A.; Nagasaki, Y. Block copolymer micelles
for drug delivery: design, characterization and biological significance.
Adv. Drug Delivery Rev. 2001, 47, 113−131.
(
(29) Yang, R.; Meng, F.; Ma, S.; Huang, F.; Liu, H.; Zhong, Z.
mitochondria in normal and cancer tissues, and its modulation.
Toxicology 2000, 155, 73−82.
(
Galactose-decorated cross-linked biodegradable poly(ethylene glycol)-
b-poly(ε-caprolactone) block copolymer micelles for enhanced
hepatoma-targeting delivery of paclitaxel. Biomacromolecules 2011,
8) Mao, Z.; Qing, Z.; Qing, T.; Xu, F.; Wen, L.; He, X.; He, D.; Shi,
H.; Wang, K. Poly(thymine)-templated copper nanoparticles as a
fluorescent indicator for hydrogen peroxide and oxidase-based
biosensing. Anal. Chem. 2015, 87, 7454−7460.
1
(
2, 3047−3055.
30) Torchilin, V. P. Micellar nanocarriers: pharmaceutical
perspectives. Pharm. Res. 2007, 24, 1−16.
31) Springsteen, G.; Wang, B. Alizarin Red S. as a general optical
(
9) Zeng, H.; Qiu, W.; Zhang, L.; Liang, R.; Qiu, J. Lanthanide
(
coordination polymer nanoparticles as an excellent artificial peroxidase
reporter for studying the binding of boronic acids with carbohydrates.
for hydrogen peroxide detection. Anal. Chem. 2016, 88, 6342−6348.
Chem. Commun. 2001, 1608−1609.
(
10) Deng, H.; Wu, G.; He, D.; Peng, H.; Liu, A.; Xi, X.; Chen, W.
(32) Springsteen, G.; Wang, B. A detailed examination of boronic
acid−diol complexation. Tetrahedron 2002, 58, 5291−5300.
(33) Sun, X.; Lacina, K.; Ramsamy, E. C.; Flower, S. E.; Fossey, J. S.;
Qian, X.; Anslyn, E. V.; Bull, S. D.; James, T. D. Reaction-based
indicator displacement assay (RIA) for the selective colorimetric and
fluorometric detection of peroxynitrite. Chem. Sci. 2015, 6, 2963−
Fenton reaction-mediated fluorescence quenching of N-acetyl-L-
cysteine-protected gold nanoclusters: analytical applications of hydro-
gen peroxide, glucose, and catalase detection. Analyst 2015, 140, 7650.
(
11) Fu, Y.; Yao, J.; Xu, W.; Fan, T.; Jiao, Z.; He, Q.; Zhu, D.; Cao,
H.; Cheng, J. Schiff base substituent-triggered efficient deboration
reaction and its application in highly sensitive hydrogen peroxide
vapor detection. Anal. Chem. 2016, 88, 5507−5512.
2
(
967.
34) Wang, L.; Qiao, J.; Li, H.; Hao, J.; Qi, L.; Zhou, X.; Li, D.; Ni,
Z.; Mao, L. Ratiometric fluorescent probe based on gold nanoclusters
and alizarin red-boronic acid for monitoring glucose in brain
microdialysate. Anal. Chem. 2014, 86, 9758−9764.
(
12) Yang, Y.; Zhao, Q.; Feng, W.; Li, F. Luminescent chemo-
dosimeters for bioimaging. Chem. Rev. 2013, 113, 192−270.
13) Kim, J. S.; Quang, D. T. Calixarene-derived fluorescent probes.
Chem. Rev. 2007, 107, 3780−3799.
14) Chinen, A.; Guan, C.; Ferrer, J.; Barnaby, S.; Merkel, T.; Mirkin,
(
(35) Billingsley, K. L.; Balaconis, M. K.; Dubach, J. M.; Zhang, N.;
Lim, E.; Francis, K. P.; Clark, H. A. Fluorescent nano-optodes for
(
glucose detection. Anal. Chem. 2010, 82, 3707−3713.
C. Nanoparticle probes for the detection of cancer biomarkers, cells,
(36) Chen, P.; Wan, L.; Ke, B.; Xu, Z. Honeycomb-patterned film
and tissues by fluorescence. Chem. Rev. 2015, 115, 10530−10574.
segregated with phenylboronic Acid for glucose sensing. Langmuir
011, 27, 12597−12605.
37) Tomsho, J. W.; Benkovic, S. J. Elucidation of the mechanism of
(
15) Kim, H. N.; Ren, W. X.; Kim, J. S.; Yoon, J. Fluorescent and
2
(
colorimetric sensors for detection of lead, cadmium, and mercury ions.
Chem. Soc. Rev. 2012, 41, 3210−3244.
the reaction between phenylboronic acid and a model diol, Alizarin
(
16) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J. Fluorescent
Red S. J. Org. Chem. 2012, 77, 2098−2106.
chemosensors based on spiroring-opening of xanthenes and related
(38) Xu, Z.; Morita, K.; Sato, Y.; Dai, Q.; Nishizawa, S.; Teramae, N.
derivatives. Chem. Rev. 2012, 112, 1910−1956.
Label-free aptasensor for adenosine using abasic site-containing DNA
and a nucleobase-specific fluorescent ligand. Chem. Commun. 2009, 42,
(
17) Liu, C.; Shao, C.; Wu, H.; Guo, B.; Zhu, B.; Zhang, X. A fast-
response, highly sensitive and selective fluorescent probe for the
ratiometric imaging of hydrogen peroxide with a 100 nm red-shifted
emission. RSC Adv. 2014, 4, 16055−16061.
6
445−6447.
(39) Xu, Z.; Yusuke, S.; Seiichi, N.; Norio, T. Signal-off and signal-on
design for label-free aptasensor based on target-induced self-assembly
and abasic site-binding ligands. Chem. - Eur. J. 2009, 15, 10375−10378.
(40) Zhang, W.; Liu, W.; Li, P.; Huang, F.; Wang, H.; Tang, B. Rapid-
response fluorescent probe for hydrogen peroxide in living cells based
on increased polarity of C-B bonds. Anal. Chem. 2015, 87, 9825−9828.
(41) Lippert, A. R.; Van De Bittner, G. C.; Chang, C. J. Boronate
oxidation as a bioorthogonal reaction approach for studying the
chemistry of hydrogen peroxide in living systems. Acc. Chem. Res.
(
18) Albers, A. E.; Okreglak, V. S.; Chang, C. J. A FRET-based
approach to ratiometric fluorescence detection of hydrogen peroxide.
J. Am. Chem. Soc. 2006, 128, 9640−9641.
(
19) Zhu, B.; Jiang, H.; Guo, B.; Shao, C.; Wu, H.; Du, B.; Wei, Q. A
highly selective ratiometric fluorescent probe for hydrogen peroxide
displaying a large emission shift. Sens. Actuators, B 2013, 186, 681−
6
86.
2
(
011, 44, 793−804.
(
20) Vikesland, P. J.; Wigginton, K. R. Nanomaterial enabled
biosensors for pathogen monitoring - a review. Environ. Sci. Technol.
010, 44, 3656−3669.
21) Alivisatos, P. The use of nanocrystals in biological detection.
Nat. Biotechnol. 2004, 22, 47−52.
22) Lim, E. K.; Kim, T.; Paik, S.; Haam, S.; Huh, Y. M.; Lee, K.
42) Biju, V.; Itoh, T.; Baba, Y.; Ishikawa, M. Quenching of
photoluminescence in conjugates of quantum dots and single-walled
carbon nanotube. J. Phys. Chem. B 2006, 110, 26068−26074.
2
(
(
Nanomaterials for theranostics: recent advances and future challenges.
Chem. Rev. 2015, 115, 327−394.
(
23) Kataoka, K.; Harada, A.; Nagasaki, Y. Block copolymer micelles
for drug delivery: design, characterization and biological significance.
Adv. Drug Delivery Rev. 2001, 47, 113−131.
I
DOI: 10.1021/acs.langmuir.7b00189
Langmuir XXXX, XXX, XXX−XXX