Ratiometric fluorescent biosensor for hyaluronidase with hyaluronan as both nanoparticle scaffold and substrate for enzymatic reaction

Article abstract of DOI:10.1021/bm500890d

Hyaluronidases (HAase) are involved in various physiological and pathological processes and have been reported as urinary marker for bladder cancer. In this study, a novel ratiometric fluorescent sensing system based on both aggregation-induced emission (

Full text of DOI:10.1021/bm500890d

Article
pubs.acs.org/Biomac
Ratiometric Fluorescent Biosensor for Hyaluronidase with
Hyaluronan As Both Nanoparticle Scaffold and Substrate for
Enzymatic Reaction
Huafei Xie, Fang Zeng,* and Shuizhu Wu*
College of Materials Science & Engineering, State Key Laboratory of Luminescent Materials & Devices, South China University of
Technology, Guangzhou 510640, China
S
* Supporting Information
ABSTRACT: Hyaluronidases (HAase) are involved in various
physiological and pathological processes and have been reported as
urinary marker for bladder cancer. In this study, a novel ratiometric
fluorescent sensing system based on both aggregation-induced
emission (AIE) and aggregation-induced quenching (ACQ) was
developed to quantitatively assess hyaluronidase level. First, a
tetraphenylethylene derivative with positive charges (TPE-2N+, typical
AIE molecule) at both ends and an anthracene derivative with positive
charge at one end (AN-N+, typical ACQ molecule) was synthesized.
These two positively charged compounds were then mixed with a
negatively charged hyaluronan (HA), which induced the aggregation of
the compounds as well as the nanoparticles formation as a result of electrostatic complexation, with TPE-2N+ acting as cross-
linking agent. The aggregation also caused the efficient quenching of the emission of AN-N+ due to ACQ effect, as well as the
fluorescence enhancement of TPE-2N+ due to AIE effect. In the presence of HAase, the enzymatic reaction led to the
degradation of HA and triggered disassembly of the nanoparticles; as a result, the emission of AN-N+ was restored and that of
TPE-2N+ was suppressed. This fluorescence variation affords the system a robust ratiometric biosensor for HAase, and the ratio
of fluorescence intensity for AN-N+ (I414) to that for TPE-2N+ (I474) can be used as the sensing signal for detecting HAase
activity. In this system, hyaluronan serves not only as the scaffold for nanoparticle formation but also as the substrate for
enzymatic reaction. This assay system is operable in aqueous media with very low detection limit of 0.0017 U/mL and is capable
of detecting HAase in biological fluids such as serum and urine. This strategy may provide a new and effective approach for
developing other enzyme assays.
Currently, a number of methods, such as physicochemical
methods, chromatographic methods, and colorimetric ap-
proaches13−17 have been used to measure HAase level.
However, most of these methods require time-consuming
preparation steps. While fluorescence technique offers distinct
advantages including, ease of use, high sensitivity, and the real-
time monitoring capability and usability in biological
samples.18−22 The reported fluorescent methods for detecting
HAase usually require substrate modification, for example,
modifications at the free carboxyl groups of HA substrate with
fluorescein or biotin, which could affect the activity of HAase.23
In addition, these fluorescent methods employ single
fluorescent intensity as the sensing signal; hence, the sensing
may well be interfered by a variety of factors including
variations in excitation intensity, emission collection efficiency,
and probe concentration.24−28 In comparison, ratiometric
fluorescent probes that adopt simultaneous recording of two
fluorescence intensity signals in the presence and absence of
1. INTRODUCTION
Hyaluronan (hyaluronic acid, HA) is a linear mucopolysac-
charide, which is highly negatively charged and can be found in
the extracellular matrix of all vertebrates and in the capsule of
some bacteria.1−3 Hyaluronidases are the extracellular matrix-
digesting endoglycosidases that predominately degrade high-
molecular-weight hyaluronan into fragments with low molec-
ular weights.4,5 Hyaluronidases are involved in a variety of
physiological and pathological processes, such as fertilization,
embryogenesis, maintenance of the elastoviscosity of liquid
connective tissues including joint synovial and eye vitreous
fluid, control of tissue hydration and water transport, wound
healing, inflammation, and tumor growth.6−10 Bladder cancer is
among the five most common malignant tumors, it is also the
second most common tumor of the genitourinary tract and the
second most common cause of death in patients with
genitourinary cancers.11,12 Recently, HAase has been reported
as urinary marker for bladder cancer, and urinary HAase level
has been employed as an assessment in early diagnosis of
bladder cancer.4,5 Due to its physiological importance, sensitive
and selective method to detect HAase level is of great
significance.
Received: June 17, 2014
Revised: July 22, 2014
Published: July 28, 2014
© 2014 American Chemical Society
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Figure 1. Schematic illustration for the assay system and its ratiometric fluorescent response to HAase.
analyte can minimize some interfering factors and allow for
more accurate detection.29−31 Therefore, for HAase detection,
there is still a high demand to improve the available probes for
utilization in biological samples with respect to their
convenience, sensitivity, ratiometric sensing, and unmodified
HA substrate.
hyaluronan (HA), and nanoparticles readily formed due to
electrostatic complexation, with TPE-2N+ acting as cross-
linking agent. Because of the aggregation, the emission of AN-
N+ was efficiently quenched due to ACQ effect, while the
emission of TPE-2N+ was greatly enhanced due to AIE effect.
In the presence of HAase, the enzymatic reaction led to the
degradation of HA, which in turn triggered the nanoparticle
disassembly; as a result, the emission of AN-N+ was restored
and that of TPE-2N+ was suppressed. The ratio of fluorescence
intensity for AN-N+ to that for TPE-2N+ can therefore be used
as the sensing signal for detecting HAase activity. Compared
with other HAase assays, this approach shows comparable or
even better sensing performance with no need for substrate
modifications (which may affect the activity of enzyme).
Additionally, due to the ratiometric sensing mechanism,
fluorescence color change during the detection process can
be easily observed by naked eyes under a hand-held UV lamp.
This assay herein was also investigated for detection of HAase
activity in biological fluid such as human serum and urine.
Ratiometric fluorescent sensing can be realized through
various mechanisms such as fluorescence resonance energy
transfer (FRET) and incorporation of a reference dye.32,33 The
preparation of these ratiometric fluorescent sensing systems
usually requires relatively complicated synthetic process. For
example, to prepare FRET-based ratiometric fluorescent
sensing, usually two fluorophores need to be incorporated
simultaneously into the sensing system, which increases
synthetic complexity.34,35 For most of the ratiometric
fluorescent sensors, a variety of common fluorophores were
used as the emissive units.36,37 These fluorophores usually
display aggregation-caused quenching (ACQ) in aqueous or
physiological media, and this is attributed to the nonradiative
relaxation of the aggregates’ excited states.38−40 Contrary to
conventional fluorophores, some organic compounds are
nonemissive in molecularly dissolved state but become highly
emissive when aggregated due to the restricted intramolecular
rotation. This unusual emission phenomenon was referred to as
‘‘aggregation-induced emission’’ (AIE).41−44
In this study, we sought to design a ratiometric sensing
system for HAase with unmodified substrate. By employing
unmodified HA as both the nanoparticle formation scaffold and
the substrate for enzymatic reaction, and making use of both
the ACQ effect and AIE effect, we fabricated a new ratiometric
biosensor for HAase. The schematic illustration for the
ratiometric fluorescent detection is shown in Figure 1. First, a
tetraphenylethylene derivative with positive charges at both
ends (TPE-2N+, typical AIE molecule) and an anthracene
derivative with positive charge at one end (AN-N+, typical
ACQ molecule) were synthesized, then these two positively
charged compounds were mixed with the negatively charged
2. EXPERIMENTAL SECTION
2.1. Materials and Reagents. Hyaluronan (molecular weight:
1000 kDa) from bovine vitreous humor, 9-(chloromethyl)anthracene,
titanium tetrachloride, potassium tetraborate, p-dimethylaminobezal-
dehdye, 4-methylbenzophenone, and bovine testicular hyaluronidase
(EC 3.2.1.35, type 1-S, 476 U/mg) were purchased from Sigma-
Aldrich, and used as received. Azobis(isobutyronitrile) (AIBN), N-
bromobutanimide (NBS), dimethyl sulfoxide (DMSO for HPLC),
N,N-dimethylformamide (DMF) and triethylamine (TEA) were
obtained from Alfa Aesar. Sodium chloride, dichloromethane, CCl4,
tetrahydrofuran (THF), acetone, diethyl ether, petroleum ether, and
other inorganic salts were analytically pure reagents. The water used in
this study was the triple-distilled water which was further treated by
ion exchange columns and then by a Milli-Q water purification system.
2.2. Synthesis of (9-Triethylammonium) Anthracene Chlor-
ide (AN-N+). The synthesis was briefly described as follows: into a 100
mL round-bottom flask fitted with a condenser were added the 9-
(chloromethyl)anthracene (226.7 mg, 1 mmol) and 20 mL of acetone,
and then triethylamine (303.57 mg, 3 mmol) was added. The mixture
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was refluxed at 60 °C for 8 h. The precipitate was filtrated and washed
by ethyl ether (3 × 20 mL). After being dried in vacuum oven, the
solid was obtained in 51.6% yield. 1H NMR (400 MHz, D2O), δ
(TMS, ppm): 8.05−7.96 (s, 1H), 7.83−7.75 (d, 2H), 7.72−7.63 (d,
2H), 7.54−7.43 (t, 2H), 7.41−7.33(t, 2H), 4.49−4.58 (s, 2H), 3.12−
2.93 (m, 6H), 0.82−0.64 (t, 9H). MS (ESI): m/z 291.1 [M]+.
2.3. Synthesis of 1,2-bis(4-Methylphenyl)-1,2-diphenyle-
thene (Compound 2). This compound was prepared according to
previously published experimental procedures.45 The synthesis was
briefly described as follows: Into a vacuum-evacuated, nitrogen-filled
250 mL two-necked, round-bottomed flask was added 3.92 g (0.02
mol) of 4-methylbenzophenone and 100 mL of THF. The solution
was cooled down to −5 °C, into which TiCl4 (5.69 g, 0.03 mol) and
Zn dust (2.62 g, 0.04 mol) were added. After being refluxed overnight,
the reaction mixture was cooled to room temperature and filtered
through a pad of silica gel. The filtrate was concentrated and the crude
product was further purified by a silica gel column using petroleum
ether as eluent. A white solid was obtained in 85.2% yield (3.06 g). 1H
NMR (400 MHz, DMSO-d6), δ (TMS, ppm): 7.15−7.07 (m, 4H),
6.98−6.91 (m, 10H), 6.86−6.82 (m, 4H), 2.21 (d, J = 5.6, 6H). MS
(ESI): m/z 360.4 [M]+.
excitation at 365 nm and the ratios of fluorescence intensity at 414 and
474 nm (I414/ I474) were calculated.
2.8. HAase Activity Detection in Biological Fluid Samples.
The urine was obtained from healthy adult men and serum was
obtained from a local hospital. For HAase detection in serum samples,
the fluorescence measurements were recorded for samples containing
the sensing system solution (final concentration: AN-N+ 4 μM/TPE-
2N+ 26 μM/HA 0.003 mg/mL) with or without HAase in PBS buffer
(pH 4.3, 0.1 mM) at 37 °C (for the samples with added HAase, the
measurements were conducted after 100 min of mixing), and the
fluorescence intensity ratios I414/I474 were calculated. The final
concentration of the serum in the test solutions is 50-fold diluted.
The determination of HAase level in human urine samples was
performed as follows: First, the urine samples were buffered at pH 4.3
using NaH2PO4, Na2HPO4, and NaCl. Then, 100 μL of each urine
sample was mixed properly with 20 mg of chitosan and then
centrifuged at 8000 rpm for 10 min (chitosan is insoluble in urine
sample and was used to agglomerate all negatively charged moieties
present in urine sample without adsorbing HAase molecules, in
accordance with literature report).16 Then, urine supernatant was
added into the probe solution, the fluorescence measurements were
conducted as mentioned previously.
2.4. Synthesis of 1,2-bis[4-(Triethylammoniomethyl)-
phenyl]-1,2-diphenylethene Dibromide (TPE-2N+). This TPE-
2N+ was prepared according to previously published experimental
procedures.46 The synthesis was briefly described as follows: Into a 25
mL round-bottom flask was added 0.36 g (1 mmol) of 2, 0.356 g (2
mmol) of freshly recrystallized NBS, and a catalytic amount of AIBN
in 10 mL of CCl4. The solution was refluxed at 80 °C for 10 h. After
being cooled to room temperature, the solution was filtered and the
filtrate was concentrated. The solvent was removed by vacuum-rotary
evaporation procedure and the crude product was obtained. Afterward,
into a 50 mL round-bottom flask fitted with a condenser were added
the crude product 3 (512 mg, 1 mmol) and 20 mL of acetone, and
then, triethylamine (607.14 mg, 6 mmol) was added. The mixture was
refluxed at 60 °C for 8 h. The precipitates was filtrated and washed by
acetone (3 × 20 mL). After being dried in vacuum oven, the white
solid was obtained in 25.6% yield. 1H NMR (400 MHz, DMSO-d6), δ
(TMS, ppm): 7.34−7.31 (m, 4H), 7.19−7.10 (m, 10H), 7.03−7.00
(m, 4H), 4.44−4.41 (s, 4H), 3.10−3.20 (m, 12H), 1.30−1.20 (m,
18H). MS (ESI): m/z 280.1 [M-2Br]2+.
3. RESULTS AND DISCUSSION
3.1. Preparation and Characterization of the Assay
System. To prepare the assay system, its two fluorophore
components, TPE-2N+ and AN-N+, were first synthesized,
respectively; the synthesis routes for the two fluorohpores were
described in Supporting Information (SI) Scheme S1. The final
products were characterized by 1H nuclear magnetic resonance
(1H NMR) and mass spectrometry (MS) (SI Figures S1−S4),
which confirmed the successful synthesis of TPE-2N+ and AN-
N+.
Due to the existence of quaternary ammonium group, AN-
N+ is well soluble in PBS buffer solution (0.1 mM, pH 4.3;
previous studies have shown that the optimal pH for HAase is
around 4.313,16,51) and shows blue emission at around 414 nm.
Upon gradual addition of HA, the fluorescence intensity of AN-
N+ at around 414 nm decreases accordingly (SI Figure S5).
This is simply because, AN-N+ molecules will bind and/or
aggregate along the HA chain due to electrostatic complexation
between HA and AN-N+, and also the ACQ effect of AN-N+;
hence, as the amount of HA is increased, the aggregation is
enhanced, corresponding the fluorescence of AN-N+ further
weakens. On the contrary, the fluorescence intensity of TPE-
2N+ in PBS buffer solution is very weak due to the lack of
aggregation and the AIE-active feature of this fluorophore;
however, the emission of the fluorophore solution at 474 nm is
greatly enhanced upon addition of HA as shown in SI Figures
S6 and S7 (the absorption spectra are shown in SI Figure S8).
The electrostatic interactions between the positively charged
TPE-2N+ and the negatively charged HA results in the
formation of nanoaggregates, and the fluorescence intensity
increases significantly due to the AIE effect.
2.5. Measurements. 1H NMR spectra were recorded on a Bruker
Avance 400 MHz NMR Spectrometer. Mass spectra were obtained
through Bruker Esquire HCT Plus mass spectrometer. Fluorescence
spectra were recorded on a Hitachi F-4600 fluorescence spectropho-
tometer with excitation wavelength being 365 nm. The particle size
and distribution was determined by dynamic light scattering (DLS) on
a Malvern Nano-ZS90 particle size analyzer at a fixed angle of 90° at
25 °C. Transmission electronic microscopy (TEM) experiments were
carried out by mounting an aqueous drop (∼15 μL) of the solution
onto a carbon-coated copper grid and observation was carried out on a
JEM-2010HR transmission electron microscopy (Japan).
2.6. Preparation of the Sensing System’S Stock Solution.
The sensing system’s stock solution was prepared by dissolving AN-N+
and TPE-2N+ and HA in buffer under stirring, and nanoaggregates
readily formed due to one-pot assembly through electrostatic
complexation between AN-N+ and TPE-2N+ and HA in buffer.
Briefly, HA was dissolved in PBS buffer (pH 4.3, 0.1 mM), and AN-N+
and TPE-2N+ solutions (with different molar ratios) were simulta-
neously added under stirring for 2 min. Then, the nanoparticle
dispersions were obtained and stored at 2−8 °C for use.
2.7. Enzyme Triggered Disassembly Process. The HAase
detection experiments were conducted as follows: 0.5 mL HAase
solution of varying concentrations was added to 2.5 mL of the sensing
system solution (nanoparticle dispersion) and the mixtures (in the
final testing solution, 4 μM AN-N+/26 μM TPE-2N+/0.003 mg/mL
HA were kept constant) were incubated at 37 °C for a certain period
of time. Termination of enzymatic reaction was achieved by heating
the mixtures in boiling water for 10 min. After cooling the mixtures to
room temperature, the fluorescence spectrum was recorded with the
In principle, the HA/AN-N+ or HA/TPE-2N+ system may be
used to detect HAase activity; as shown in SI Figures S5 and
S6, it is clear that the system consisting of only one probe
(either AN-N+ or TPE-2N+) and HA can also function as the
detection system for HAase; however, these two systems can
only function either as the Turn-ON or Turn-OFF sensors for
HAase. While ratiometric fluorescent sensing method depends
on simultaneous variation of fluorescence intensities at two
different wavelengths, thus providing a built-in self-calibration
and higher accuracy for quantitative analysis.47
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Figure 2. (A) Size distribution of the sensing system in PBS buffer solution (pH 4.3) (4 μM AN-N+/26 μM TPE-2N+/0.003 mg/mL HA)
determined by DLS; (B) TEM image of the sensing system.
3.2. Fluorescent Response of the Sensing System
toward HAase. To investigate the biosensor’s fluorescent
response toward HAase, we measured the fluorescent spectra of
the sensing system in the absence or presence the different
concentrations of HAase. The fluorescence spectra of the
system were periodically recorded during incubation of the
assay system with HAase at 37 °C in PBS buffer (0.1 mM, pH
4.3).
To determine the suitable AN-N+/TPE-2N+ ratio for
preparing the sensing system, we first measured the
fluorescence spectra of the ensemble with different molar
ratios by keeping the concentration of HA constant at 0.003
mg/mL and the amount of HAase at 15 U/mL (SI Figures S9).
When the AN-N+/TPE-2N+ ratio is in the range 4:26−7:20,
Figure 3. Plot of the fluorescence intensity I414/I474 of the sensing
system (4 μM AN-N+/26 μM TPE-2N+/0.003 mg/mL HA) versus
reaction time in the presence of different amounts of HAase and the
denatured enzyme in PBS buffer solution (pH 4.3) (λex = 365 nm).
the fluorescence intensities of two fluorophores in the presence
or absence of HAase are comparable, and in the following
HAase detection experiments, we adopted the ratio of 4:26. It is
clear, as the enzymatic reaction time is increased, the
fluorescence intensity at 474 nm decreased gradually
simultaneously with a gradual increase of emission at 414
nm; and there exists a clear isosbestic point.
The photobleaching of the sensing system in PBS buffer was
also investigated. Despite direct exposure to UV lamps (254
and 365 nm simultaneously) for 0 to 180 min, the fluorescence
spectra and the emission intensity ratio I414/I474 of the sensing
system show only slight change (SI Figure S10), this indicates
that the nanoaggregates formed by AN-N+/TPE-2N+/HA are
stable and have very good photostability. Moreover, we studied
the nanoaggregates using DLS and TEM. The formation of
spherical nanoaggregates with a diameter in the range 80−100
nm has been observed by TEM, as shown in Figure 2, this size
is smaller than that (∼170 nm) determined by the DLS.
The kinetic activity of the enzymatic reaction was determined
by measuring the time-dependent fluorescence changes of the
sensing system (4 μM AN-N+/26 μM TPE-2N+/0.003 mg/mL
HA) in the presence of different concentrations of HAase, as
shown in Figure 3 and Figure 4. In the absence of HAase, we
did not notice any changes in the fluorescence spectrum, which
also proves the stability of the system. The time-course
experiments show that the emission intensity ratio of I414/I474
gradually increases as enzyme concentration and time increase,
and reaches a plateau after 85 min of reaction time when HAase
level is kept at 10 U/mL. To ensure complete reaction, the
incubation time of 100 min was employed in all other
experiments. Fluorescence color change of the sensing system
in the absence of HAases and in the presence of different
HAase levels can be clearly observed form blue to greenish blue
under a hand-held UV lamp (SI Figure S11).
Figure 4. Time course of fluorescence spectra of the sensing system (4
μM AN-N+/26 μM TPE-2N+/0.003 mg/mL HA) in the presence of
HAase. The measurements were conducted with HAase (15 U/mL) in
PBS buffer solution (pH 4.3, 0.1 m). Spectra measured every 5 min are
displayed (λex = 365 nm).
3.3. Detection Limit of the Biosensor. To demonstrate
the efficiency of this sensing system in the measurement of
HAase, various HAase levels (0.005−10 U/mL) were added to
the sensing system solutions (4 μM AN-N+/26 μM TPE-2N+/
0.003 mg/mL HA), and then, the fluorescent spectra were
measured after 100 min incubation. As shown in Figure 5, the
fluorescent intensities at 414 nm increase, while those at 474
nm decrease with increasing levels of HAase. The fluorescence
intensity ratio of I414/I474 were linearly related to the HAase
level at the low concentration range of 0.005−0.2 U/mL
(2.38−95.2 mg/mL; SI Figure S12). The detection limit was
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Figure 5. (A) Fluorescence spectra of the sensing system (4 μM AN-N+/26 μM TPE-2N+/0.003 mg/mL HA) in the absence of HAase and in the
presence of different HAase levels in PBS buffer solution (0.1 mM, pH 4.3). (B) Fluorescence intensity ratio of I414/I474 for the sensing system (4
μM AN-N+/26 μM TPE-2N+/0.003 mg/mL HA) in PBS buffer solution (0.1 mM, pH 4.3) as a function of HAase level (λex = 365 nm).
determined to be as low as 0.0017 U/mL (0.81 mg/mL). These
results show that this sensing system is sensitive to
hyaluronidase activity. Since HAase level in normal human
the positively charged TPE-2N+ and AN-N+ can no longer form
nanoaggregates, and both fluorophores are water-soluble; as a
result, the AIE effect of TPE-2N+ and the ACQ effect of AN-N+
are turned off. To further confirm that the nanoaggregate
disassembly is triggered by the enzymatic reaction of HAase, we
heated the nanoaggregate sensing system up to 100 °C without
the addition of HAase and then measured the size of the
nanoparticles by DLS, as shown in SI Figure S13. It can be seen
that, without addition of HAase, the nanoaggregate size
basically remains unchanged, which again proves the dis-
assembly is caused by HAase.
serum is 0.256
0.198 U/mL, and HAase level in normal
human urine is 0.0042 0.0012 U/mL,48 with the detection
limit of 0.0017 U/mL, this system has the potential to be used
for HAase level determination in biological fluids samples.
The sensing mechanism involves nanoparticle formation
between the two positively charged fluorophores (the AIE-
active TPE-2N+ and the ACQ-active AN-N+) and the
negatively charged HA, as well as the nanoaggregate
disassembly triggered by enzymatic reaction of HAase. To
further confirm the sensing mechanism, we studied the
nanoaggregate formation and disassembly triggered by
enzymatic reaction by TEM and DLS measurements. As
shown in Figures 6 and 7, in the absence of HAase, the size of
3.4. HAase Detection in Biological Fluid Samples.
Based on the above results, we set out to evaluate the efficacy of
this assay system in real biological samples. It was reported that
high urinary and serum levels of HAase have been used for
detection of high-grade bladder carcinoma,49,50 so we measured
HAase levels in the urine and serum with different levels. In this
study, the HAase level of each sample was determined from the
standard curve (SI Figure S12) and listed in SI Table S1 and
Table S2. For urine samples, chitosan, which is insoluble in
urine sample, was used to agglomerate all negatively charged
moieties present in urine sample without adsorbing HAase
molecules (HAase has relatively low molecular weight),
according to literature-reported method.16 The endogenous
(originally existing) HAase in urine and diluted serum sample
was determined by the system and the traditional colorimetric
Morgan−Elson assay method51 for comparison. As can be seen
from the results, the deviation between the two assay methods
is less than 10%, and the endogenous HAase levels for the urine
and undiluted serum sample are then calculated as 5.12 × 10−3
U/mL and 0.317 U/mL, respectively. In addition, the possible
interference of biologically relevant species (including salts,
vitamins, and amino acids) was also investigated, and the results
are shown in SI Figure S14. It is obvious that only HAase
induces a prominent fluorescence change, whereas the addition
of other species under the same conditions leads to almost no
fluorescence change. These results, in combination with the
unique specificity of the enzymatic reaction, confirm that the
system has good selectivity toward HAase over other
biologically relevant species.
Figure 6. Size distribution of the nanoaggregates determined by
dynamic light scattering (DLS) upon addition of different amounts of
HAase.
the nanoparticles formed by the sensing system is around 170
nm by DLS and 80−100 nm by TEM. Upon addition of HAase,
the enzymolysis leads to the degradation of HA and
corresponding disassembly of the nanoparticles. With the
increased HAase levels, the size of the nanoparticles decreases
gradually. Similar results were also obtained by TEM (Figure
7). In the presence of HAase, HA is degraded into fragments
with small molecular weights; hence, the electrostatic
interactions between these negatively charged fragments and
Furthermore, the sensing system has also been successfully
used to detect the HAase levels in urine and diluted serum
samples spiked with HAase in this study. The recovery of added
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Figure 7. HR-TEM images of the assay systems with the addition of HAase (A, 0; B, 0.25; C, 0.5; D, 1.0 U/mL, respectively) placed on copper grids.
Scale bar: 100 nm.
known amounts of HAase into the urine and serum samples are
in the range 97.55−102.15% and 98.95−108.92%, these results
suggest the accuracy and reliability of the present method for
HAase determination, and this system could be used for
diagnosis of bladder carcinoma. The sensing system’s response
toward HAase is repeatable and reproducible.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support by the
National Key Basic Research Program of China (Project No.
2013CB834702), and NSFC (21025415 and 21174040).
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4. CONCLUSIONS
In summary, we successfully designed a fluorescent ratiometric
biosensor for HAase through employing unmodified HA as the
nanoparticle formation scaffold and the substrate for enzymatic
reaction and by using the ACQ effect and AIE effect. Compared
with other HAase assays, this approach shows comparable or
better performance with no need for substrate modifications
which may affect the activity of enzyme. Moreover, due to the
ratiometric sensing mechanism, higher sensitivity and reliability
can be achieved and the fluorescence color change during the
detection process can be easily observed by naked eyes under a
hand-held UV lamp. This assay herein can be used detection of
HAase level in biological fluid such as human serum and urine.
This strategy may offer a technically simple approach for
designing ratiometric fluorescent sensing systems for detecting
other biomolecules in biological samples.
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* Supporting Information
1
Synthetic routes, H NMR spectra, mass spectra, fluorescence
spectra, absorption spectra, photographs, determination of the
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HAase level in serum, and detection of HAase level in urine.
This material is available free of charge via the Internet at
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