chemosensors
Article
Hierarchical Self-Assembly of Amino Acid
Derivatives into Enzyme-Responsive
Luminescent Gel
Yibao Li *, Yu Peng, Wei Liu, Yulan Fan, Yongquan Wu, Xun Li * and Xiaolin Fan
Key Laboratory of Organo-Pharmaceutical Chemistry, Gannan Normal University, Ganzhou 341000, China;
[email protected] (Y.P.); [email protected] (W.L.); [email protected] (Y.F.);
[email protected] (Y.W.); [email protected] (X.F.)
* Correspondence: [email protected] (Y.L.); [email protected] (X.L.); Tel.: +86-797-839-3536 (Y.L.)
Academic Editor: Manel Del Valle
Received: 15 November 2016; Accepted: 25 January 2017; Published: 7 February 2017
Abstract: In this study, a novel three-component hydrogel has been designed and fabricated via
hierarchical self-assembly by amino acid derivative (N,N 0 -diphenylalanine-3,4,9,10-tetracarboxylic
diimide (NPPD)), riboflavin (RF) and α-cyclodextrin (α-CD). These molecules were aggregated to
form some fibrous structures based on hydrogen bond and π–π stacking. The results show that the
hydrogel has a specific response to α-amylase and the fluorescence disappears once hydrolyzed.
Therefore, this multi-component hydrogel has potential application in the field of drug delivery.
Keywords: hydrogel; amylase-response; hydrogen bond; drug delivery
1. Introduction
The stimuli-responsive hydrogels, responsive to subtle changes in ambient systems, are of great
interest to researchers; their use ranges from physical triggers to chemical triggers due to their wide
application in drug delivery, sensors, and molecular recognition [1]. Another potential application is
as the pH-responsive luminescent gel display in nano pH sensors [2]. The stimuli-responsive methods
include temperature, pressure, pH, concentration, enzyme, light, etc. [3–6]. Among the above different
stimuli-responsive methods, enzymes have great advantages. The enzyme is a substrate-specific,
highly efficient catalytic under mild conditions and is associated with disease [7–11]. The biology
has a vast enzymatic stimuli-responsive material because all processes are ultimately controlled by
enzymes in living organisms [9]. Thus, it is necessary to design and fabricate new stimuli-responsive
materials. Furthermore, these self-assembled nanostructures could be designed with novel peptide
motifs [12–14].
Recently, the enzyme-responsive systems fabricated by non-covalent interactions have attracted
more and more attention [15–23]. As one of the non-covalent interactions, π–π stacking is vital
to accelerate the alliance of supramolecular compounds to provide self-assembled structures
with high stability [24]. Up to now, a lot of research for enzyme-responsive systems has been
reported [25–29]. For instance, Zhang and coworkers have constructed several polymers by the
electrostatic interaction between components responsive to phosphatase or acetylcholine [30–33].
In general, most enzyme-responsive materials consist of two components: (i) an enzyme sensitive
component, which is a substrate or substrate mimic based on a biomolecule (such as a peptide, a lipid
or a polynucleotide); (ii) a component that directs and controls changes in non-covalent interactions [8].
Compared with covalently modifying the building blocks, utilizing the non-covalent bond to build
the enzyme-response system has some advantages. For example, it is easier to modify or regulate the
function of the system and it reduces the difficulty of synthesis and purification.
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In this work, we have attempted to fabricate an amylase response gel using a non-covalent
bond between amino acid derivatives and guest molecules. As is shown in Figure 1, the amino acid
derivative (NPPD) was synthesized by 3,4,9,10-perylenetetracarboxylic dianhydride and phenylalanine
via amino bond. The phenylalanine with an aromatic ring can affect the morphology of the assemblies
and reduce the minimal association concentrations [34–38]. In addition, the perylene is a classic organic
chromophore with strong π–π stacking. A compound with a chromophore could form hydrogel, which
may have advantageous photonic and electronic characteristics [39]. Riboflavin with the conjugated
aromatic moieties, named flavin nucleotides, behaves as a photoreceptor in the phototropism of plants,
plays a significant role in various biochemical processes in plants and also acts as a perfect component
in hydrogel systems [40]. As a guest molecule, the α-cyclodextrin (α-CD) plays a key role in the
specific response of the α-amylase. Moreover, the α-CD molecules can form four hydrogen bonds,
because there is a glucose unit in the position of the distortion. The three compounds could form
hydrogel under ultrasound at low temperature (~15 ◦ C). When the α-amylase was added at 37 ◦ C,
the hydrogel collapsed.
2. Materials and Methods
2.1. Materials
Anhydrous ethanol, imidazole, L-phenylalanine, perylene-3,4,9,10-tetracarboxylic dianhydride
were purchased from J & K Scientific Ltd. (Beijing, China). All chemical reagents were used without
any further processing. The N,N 0 -diphenylalanine-3,4,9,10-tetracarboxylic diimide (NPPD) was
synthesized and characterized in the laboratory.
2.2. Synthesis of NPPD
An amount of 0.392 g (1.0 mmol) perylene-3,4,9,10-tetracarboxylic dianhydride, 0.330 g (2.0 mmol)
L-phenylalanine and 2.0 g imidazole were heated at 90 ◦ C for 40 min under nitrogen atmosphere. When
the imidazole was melted, the temperature was raised to 120 ◦ C for 6 h. Then, 25 mL of CH3 CH2 OH
was poured into the round-bottom flask, refluxed for 6 h at 90 ◦ C, cooled down at room temperature,
then filtered by vacuum suction and washed with CH3 CH2 OH. The sediment was dissolved by H2 O.
Then, 1.0 moL/L HCl was dropped into the solution until the mixture solution was acidic. The solution
was filtered by vacuum suction and washed with H2 O in ambient temperatures. The product was
dried at room temperature to obtain a violet powder (yield: 0.57 g, 87%). The structure and purity
of the product were confirmed by IR (KBr): 1252.4, 1396.4, 1501.2 cm−1 (C=C), 1344.9, 1432.5 cm−1
(-CH2-), 1656.6, 1697.0, 1737.9 cm−1 (C=O), 2934.8 cm−1 (C-H), 3439.3 cm−1 (O-H); 1 H NMR (DMSO,
400 MHz, ppm): 8.31–8.45 (q, 8H), 7.13–7.23 (m, 8H), 7.04–7.08 (t, 2H), 5.92–5.95 (q, 2H), 3.59–3.64 (q,
2H), 3.40–3.44 (t, 2H), 3.32 (s, 2H); MALDI-TOF-MS: calcd for C42 H28 N2 O8 , 686.1.
2.3. Characterization
Ultraviolet (UV) absorption spectra were obtained using a UV-2700 UV-Vis spectrophotometer
and NMR spectra were measured on a Bruker ULTRASHIELD 400 (1H NMR 400 MHz) spectrometer.
The Fourier transform-infrared spectroscopy (FT-IR) were recorded on an AVATAR 360 FT-IR
spectrophotometer, the powder samples were mixed with KBr to prepare the thin films in the solid-state
FT-IR studies. The luminescence spectra were measured on a LS55 fluorescence spectrophotometer,
in which the path length of the quartz cell is 1 cm. The emission bandwidth was 5 nm. SEM images
of these samples were recorded using FEI QUANTA 450 with an accelerating voltage of 25.0 kV.
The samples were obtained by dropping the gels on a cylindrical aluminum substrate and drying at
room temperature. Confocal laser scanning microscope (CLSM) images were taken with a confocal laser
scanning fluorescent microscope FV 1000 invert microscope (OLYMPUS) under 100× magnifications
at an excitation wavelength of 405 nm; the samples were coated with gold using a MSP-1S magnetron
sputter (Japan) coater.
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2.4.
2.4. Gelation
Gelation Preparation
Preparation
The
NPPD/α-CD/RF= 1:1:2
1:1:2was
was
a capped
glass
bottle
2.0 =mL
The NPPD/α-CD/RF=
putput
into ainto
capped
glass bottle
(5 mL)
with(52.0mL)
mL Vwith
4:6
THF :VH2O
◦ C.
V
THF:VH2Osolvent.
= 4:6 mixture
solvent.
Then,
hydrogel
was
obtained
after ultrasonic
treatment
mixture
Then, the
hydrogel
wasthe
obtained
after
ultrasonic
treatment
for 40 min
at 15–20 for
40
min
at 15–20
°C. The
critical gelation
concentration
(CGC) was 0.6% w/v.
The
critical
gelation
concentration
(CGC)
was 0.6% w/v.
2.5. α-Amylase
α-AmylaseResponsive
ResponsiveTest
Test
2.5.
An amount
amount of 200 µL
min
at
An
μL α-amylase
α-amylase(50
(50U/mL)
U/mL) was
was added
addedinto
intothe
thehydrogel,
hydrogel,and
andheated
heated5–10
5–10
min
3737 °C.
The
α-CD
was
gradually
hydrolyzed
byby
thethe
α-amylase,
and
thethe
gelgel
eventually
collapses.
at
The
α-CD
was
gradually
hydrolyzed
α-amylase,
and
eventually
collapses.
◦ C.
3. Results
Resultsand
and Discussion
Discussion
3.
The chemical
chemical structure
shown
in in
Figure
1, respectively.
TheThe
NPPD
has
The
structure of
ofNPPD,
NPPD,RF,
RF,and
andα-CD
α-CDare
are
shown
Figure
1, respectively.
NPPD
beenbeen
synthesized
and characterized
as is shown
in Supplementary
MaterialsMaterials
Figure S1.Figure
The NPPD
has
has
synthesized
and characterized
as is shown
in Supplementary
S1. The
a
perylene
core
and
two
L
-phenylalanine
residues.
A
number
of
π
electrons
or
hydrogen
bonding
sites
NPPD has a perylene core and two L-phenylalanine residues. A number of π electrons or hydrogen
are contributing
form hydrogel.
has hydroxyls
as ishas
shown
in Figure
bonding
sites aretocontributing
to The
formα-cyclodextrin
hydrogel. The(α-CD)
α-cyclodextrin
(α-CD)
hydroxyls
as 1b,
is
which
could
form
hydrogen
bonding
with
the
NPPD
or
RF.
Moreover,
this
is
a
specific
response
shown in Figure 1b, which could form hydrogen bonding with the NPPD or RF. Moreover, this is to
a
α-amylase.
When the
ratio ofWhen
NPPD/RF/α-CD
1/1/2,
three-component
is
specific
response
to molar
α-amylase.
the molar isratio
of the
NPPD/RF/α-CD
is hydrogel
1/1/2, the
formed under ultrasound
temperature
(Figure S2).
three-component
hydrogelatisroom
formed
under ultrasound
at room temperature (Figure S2).
0 -diphenylalanine-3,4,9,10-tetracarboxylic diimide (NPPD);
Figure 1.
1. Chemical
Chemicalstructures
structuresofofN,
N,N
Figure
N′-diphenylalanine-3,4,9,10-tetracarboxylic
diimide (NPPD);
α-cyclodextrin (α-CD);
(α-CD); riboflavin
riboflavin (RF).
(RF).
α-cyclodextrin
In
ordertotoexplore
explore
molecular
interactions
gel, the fluorescence
spectrum,
In order
the the
molecular
interactions
of the of
gel,the
the fluorescence
spectrum, ultraviolet
ultraviolet
spectrum
infraredwere
spectrum
wereThe
recorded.
The spectrum
fluorescence
spectrum of
spectrum and
infraredand
spectrum
recorded.
fluorescence
of NPPD/α-CD/
NPPD/α-CD/RF=
1:1:2
in hybrid
solution
(V4/6,
THF/V
H2O = 4/6,
[NPPD]
× 10−6was
mol/L)
was
RF = 1:1:2 in hybrid
solution
(VTHF
/VH2O =
[NPPD]
= 1.0
× 10−=6 1.0
mol/L)
shown
in shown
Figure in
2a.
Figure
2a. The absorption
peak
the
gel and
NPPD
were at
observed
in the fluorescence
The absorption
peak of the
gel of
and
NPPD
were
observed
538 nmat
in538
the nm
fluorescence
spectrum.
−6 mol/L,
spectrum.
the fluorescence
intensity
NPPD molecules
= 1.0
× 10−6 mol/L,
the
However, However,
the fluorescence
intensity of
NPPD of
molecules
([NPPD] ([NPPD]
= 1.0 × 10
the black
−
6
black
line)greater
was greater
the three-component
gel ([NPPD]
= 1.0
× 10−6mol/L,
mol/L, the
the blue line) in
line) was
than than
the three-component
gel ([NPPD]
= 1.0
× 10
in the
the
mixture
THF/V
H2O
= 4/6).
According
to to
thethe
previous
mixture solution
solution (VTHF
/V
= 4/6).
According
previousstudy,
study,the
thefluorescence
fluorescenceintensity
intensity of
of
H2O
perylene-diimide
perylene-diimide derivatives
derivativeswas
was enhanced
enhanced at
at low
low concentrations,
concentrations, while
while the
the fluorescence
fluorescence intensity
intensity
tended
weakened or
orquenching
quenchingunder
underthe
theaggregation
aggregation
solid
state
[41–44].
This
means
tended to be weakened
oror
solid
state
[41–44].
This
means
thatthat
the
the
assembly
aggregation
thecomponents
three components
has The
changed.
The π–π
stacking
of the
assembly
aggregation
of theof
three
has changed.
π–π stacking
of the
aggregation
is
aggregation
stronger
thanThe
thatstronger
of NPPDs.
The stronger
π–πmake
stacking
effect
will make
partial
stronger thanisthat
of NPPDs.
π–π stacking
effect will
partial
quenching
fluorescence
quenching
fluorescence
intense.
When theisexcitation
wavelength
is 490
nm, the
more intense.
When themore
excitation
wavelength
490 nm, the
fluorescence
intensity
of fluorescence
NPPD/RF is
intensity
of NPPD/CD
NPPD/RF isand
lower
than
NPPD/CD
and
NPPD (Figure
We
conjecture
the π–π
lower than
NPPD
(Figure
2b). We
conjecture
that the2b).
π–π
stacking
effectthat
between
RF
stacking
effect
betweenthan
RF and
is stronger
thatself-assemble
of NPPD. NPPDs
RF self-assemble
and NPPD
is stronger
thatNPPD
of NPPD.
NPPDsthan
and RF
by theand
hydrogen
bond and
by
hydrogen
bond in
and
π–πsolution
stackingwith
interaction
in mixed
solution with
π–πthe
stacking
interaction
mixed
the assistance
of ultrasound.
Then, the
fourassistance
hydroxyls of
of
ultrasound.
fourperipheral
hydroxylshydroxyl
of RF interacted
the peripheral
hydroxyl
via
RF interactedThen,
with the
of CDs via with
hydrogen
bonding. The
smallerof
RFCDs
is easier;
hydrogen
bonding.
The smaller
RF is to
easier;
it enters
intoπ–π
thestacking.
aggregation of NPPD to generate
it enters into
the aggregation
of NPPD
generate
stronger
stronger π–π stacking.
The UV-Vis spectra of the hydrogel dilute solution were explored, as shown in Figure 2c. The
ultraviolet absorption peak maxima of the NPPDs’ solution ([NPPD] = 1.0 × 10−5 mol/L) was
observed at 371 nm, 460 nm, 490, and 526 nm, respectively. Furthermore, the absorption peaks of
Chemosensors 2017, 5, 6
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The UV-Vis spectra of the hydrogel dilute solution were explored, as shown in Figure 2c.
The ultraviolet absorption peak maxima of the NPPDs’ solution ([NPPD] = 1.0 × 10−5 mol/L) was
Chemosensors 2017, 5, 6
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observed at 371 nm, 460 nm, 490, and 526 nm, respectively. Furthermore, the absorption peaks of RF
−5
([RF]RF
= 2.0
× =10
existed
atat350
inthe
theabsorption
absorption
peak
maximum
the gel,
([RF]
2.0 ×mol/L)
10−5 mol/L)
existed
350nm.
nm.However,
However, in
peak
maximum
of theofgel,
Chemosensors 2017, 5, 6
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a slight
change
appeared;
the
absorption
peak
at
350
nm
and
460
nm
were
shifted
respectively.
a slight change appeared; the absorption peak at 350 nm and 460 nm were shifted respectively. The The
at nm
350
nm
of −5the
gel
manifested
asomewhat
somewhat
red-shift.
was
ascribed
to maximum
the
geometry
of gel,
theof the
peak peak
at 350
of×the
gel
manifested
red-shift.
was
ascribed
to the
geometry
RF
([RF]
= 2.0
10
mol/L)
existed ata350
nm. However,
in theItIt
absorption
peak
of the
aromatic
systems
stacking
between
NPPDand
and
RF
and
thethe
NPPD/α-CD/RF
system.
aromatic
systems
stacking
between
NPPD
RF
theaggregation
aggregation
of
NPPD/α-CD/RF
system.
a slight
change
appeared;
the absorption
peak
atand
350 the
nm
and
460 nmof
were
shifted
respectively.
The
peak at 350 nm of the gel manifested a somewhat red-shift. It was ascribed to the geometry of the
aromatic systems stacking between NPPD and RF and the aggregation of the NPPD/α-CD/RF system.
Figure
(a)fluorescence
The fluorescence
of the hydrogel
and one-component
room
Figure
2. (a)2.The
spectraspectra
of the hydrogel
and one-component
solutionsolution
at roomattemperature
−6 mol/L, [α-CD]
−6 mol/L, [RF] = 2 × 10
−6
−
6
−
6
−
6
temperature
([NPPD]
=
10
=
10
mol/L);
(b)
The
fluorescence
([NPPD] = 10
mol/L, [α-CD] = 10
mol/L, [RF] = 2 × 10
mol/L); (b) The fluorescence
−6 mol/L, [α-CD]
spectra
of
andtwo-component
two-component
at room
temperature
([NPPD]
= 10
6 mol/L,
spectra
of the
NPPD
at room
([NPPD]
= 10
Figure
2. the
(a)NPPD
The and
fluorescence
spectra solution
ofsolution
the hydrogel
andtemperature
one-component
solution
at −room
−6
−6
=temperature
10 mol/L,
[RF]
=
2
×
10
mol/L);
(c)
UV-Vis
spectra
of
NPPD/α-CD/RF
=
1:1:2
system
at
room
−6
−6
−6
−
6
−
6
([NPPD]
= 10
= 10(c) mol/L,
= 2 × of
10 NPPD/α-CD/RF
mol/L); (b) The fluorescence
[α-CD] = 10 mol/L,
[RF]
= 2 mol/L,
× 10 −5[α-CD]
mol/L);
UV-Vis[RF]
spectra
= 1:1:2 system
temperature
= [α-CD]
= 10 M and−[RF]
2.0room
×10−5 temperature
M, [path length = 2.0 mm].
−6 mol/L, [α-CD]
5 M =and
spectra
of the([NPPD]
NPPD
and
two-component
at
= 10
at room
temperature
([NPPD]
= [α-CD] = 10solution
[RF] = 2.0 ×10−5([NPPD]
M, [path
length
= 2.0 mm].
= 10−6 mol/L, [RF] = 2 × 10−6 mol/L); (c) UV-Vis spectra of NPPD/α-CD/RF = 1:1:2 system at room
The infrared spectrum was recorded
to discuss the driving force of gel factors, especially for
temperature ([NPPD] = [α-CD] = 10−5 M and [RF] = 2.0 ×10−5 M, [path length = 2.0 mm].
The
infraredofspectrum
was
recorded
to discuss
the driving In
force
gel factors,
especially
for the
the presence
a hydrogen
bonding
system
by the components.
the of
figure,
the strong
C=O bond
−1 and 1657 cm−1 in pure NPPD was observed. However, the shift of
of
tertiary
amides
at
1697
cm
presence of
hydrogen
bonding
by to
thediscuss
components.
In force
the figure,
strong
C=O bond
Thea infrared
spectrum
wassystem
recorded
the driving
of gel the
factors,
especially
for of
1 and 1657
−1S5)
C=O
stretching
to
lower
(Figure
the participation
the However,
H-bond
in the
the
gel
state.
tertiary
atof1697
cm−frequency
cm
inbyattests
pure
NPPD
was observed.
shift
of C=O
theamides
presence
a hydrogen
bonding
system
the components.
In theoffigure,
the strong
C=O
bond
The
topography
of at
the1697
gel cm
was
observed
by −1scanning
electron
microscopy
(SEM).
Asthe
isthe
shown
in
−1 and
of tertiary
amides
1657attests
cm
in
pure
NPPD was
observed.
However,
of The
stretching
to lower
frequency
(Figure
S5)
the
participation
of
the H-bond
in
gelshift
state.
Figure
3, the dried
hydrogel
formed
rod-like
structures.
The three-component
xerogel
was
C=O
stretching
to
lower
frequency
(Figure
S5)
attests
the
participation
of
the
H-bond
in
the
gel
state.
topography of the gel was observed by scanning electron microscopy (SEM). As is shown in Figure 3,
composed
of a close-knit
structure,
in which
structures
a diameter
ranging
from
0.3 to
The topography
of the gel
was observed
byrod-like
scanning
electron with
microscopy
(SEM).
As is
shown
in of a
the dried
hydrogel formed
rod-like
structures.
The three-component
xerogel
was
composed
0.8
μm cross-linked
with
each other
to form
three-dimensional
(Figure 3a,b). Additionally,
Figure
3, the dried
hydrogel
formed
rod-like
structures. networks
The three-component
xerogel was
close-knit
structure,
in which
rod-like structures
with athe
diameter
ranging
fromand
0.3 stop
to 0.8solvent
µm cross-linked
the
rod-like
crosslinking
other rod-like
make
pores small
from
composed
ofbundles
a close-knit
structure,each
in which
structures
withenough
a diameter
ranging
from 0.3
to
with flowing
each other
to
form
three-dimensional
networks
(Figure
3a,b).
Additionally,
the
rod-like
bundles
which makes
theeach
hydrogel
stable.
addition, we have
done the
confocal
laser
scanning
0.8 μm cross-linked
with
other to
form In
three-dimensional
networks
(Figure
3a,b).
Additionally,
crosslinking
eachbundles
other make
the pores
enough
andgel
stop
solvent
from flowing
which
microscope
experiment,
andsmall
found
that the
is small
fluorescigenic.
(Figure
S6a,b).makes
The the
the rod-like (CLSM)
crosslinking
each
other make
the pores
enough and
stop solvent
from
hydrogel
stable.
In
addition,
havestructures
done
theIn
confocal
laser
experiment,
fluorescence
morphology
ofhydrogel
these
upon
excitation
at
405done
nm microscope
was
with scanning
the
SEM
flowing
which
makes thewe
stable.
addition,
we scanning
have
the consistent
confocal(CLSM)
laser
images,
which
demonstrated
that
the
hydrogel
was
a
luminescent
gel
with
fluorescence.
and found
that
the
gel
is
fluorescigenic.
(Figure
S6a,b).
The
fluorescence
morphology
of
these
structures
microscope (CLSM) experiment, and found that the gel is fluorescigenic. (Figure S6a,b). The
upon fluorescence
excitation atmorphology
405 nm wasofconsistent
with the
SEM
images,
which
that the SEM
hydrogel
these structures
upon
excitation
at 405
nm demonstrated
was consistent with
which demonstrated
that the hydrogel was a luminescent gel with fluorescence.
was aimages,
luminescent
gel with fluorescence.
Figure 3. The SEM images of the hydrogel ([NPPD] = 10−3 mol/L, [α-CD] = 10−3 mol/L,
[RF] = 2 × 10−3 mol/L) (a) the xerogels in a mixed solution for a larger scale; (b) NPPD/RF/α-CD
hydrogel
a mixed
solution
forof
a small
3
Figure 3.in The
SEM
images
the scale.
hydrogel ([NPPD] = 10−−33 mol/L, [α-CD] = 10−3 −
mol/L,
Figure 3. The SEM images of the hydrogel ([NPPD] = 10
mol/L, [α-CD] = 10
mol/L,
−3 mol/L) (a) the xerogels in a mixed solution for a larger scale; (b) NPPD/RF/α-CD
−
3
[RF]
=
2
×
10
[RF] = 2 × 10 mol/L) (a) the xerogels in a mixed solution for a larger scale; (b) NPPD/RF/α-CD
To explore the enzyme-responsive function of the hydrogel, a series of characterizations were
hydrogel
in a mixed
solution
a smallscale.
scale.
hydrogel
in a mixed
solution
forfor
a small
performed. The fluorescence spectrum of the hydrogel after α-amylase processing was recorded
To explore the enzyme-responsive function of the hydrogel, a series of characterizations were
performed. The fluorescence spectrum of the hydrogel after α-amylase processing was recorded
Chemosensors 2017, 5, 6
5 of 8
To explore the enzyme-responsive function of the hydrogel, a series of characterizations were
performed. The fluorescence spectrum of the hydrogel after α-amylase processing was recorded
Chemosensors 2017, 5, 6
5 of 8
(Figure 4a). Compared with the fluorescence gel, the fluorescence intensity of the gel after enzyme
treatment
decreased
a lot. with
Thethe
fluorescence
intensity
of the hydrogel
after
α-amylase
processing
(Figure
4a). Compared
fluorescence gel,
the fluorescence
intensity of
the gel
after enzyme
treatment
decreased
Thetwo-component
fluorescence intensity
of the (the
hydrogel
after
α-amylase
processing
is almost
the same
as thea lot.
pure
solution
black
line).
In addition,
theispure gel
almost
the
same
(theWhen
black line).
In addition, was
the pure
Chemosensors
2017, 5,as
6 the pure two-component
5 ofgel
8 into the
performance
of α-amylase-responsive
gel was solution
recorded.
the α-amylase
added
performance of α-amylase-responsive
gel was recorded. When the α-amylase was added into the
◦
hydrogel after 10 min at 37 C, the hydrogel collapsed. The result is shown in the optical images of
hydrogel
10 min at with
37 °C,the
thefluorescence
hydrogel collapsed.
The result is
shown of
in the
the gel
optical
(Figure after
4a). Compared
gel, the fluorescence
intensity
afterimages
enzymeof
Figure Figure
4b.
This
result
is due
toThe
the
α-CD
gelhydrolyzed
hydrolyzed
α-amylase.
This means
treatment
decreased
a lot.
fluorescence
intensity
of the hydrogel
after
α-amylase
processing
is that the
4b. This
result
is
due
to
the
α-CD in
in the
the
gel
by by
α-amylase.
This means
that the
hydrogel
has
the
enzyme-responsive
function
of
α-amylase.
almost
the
same
as
the
pure
two-component
solution
(the
black
line).
In
addition,
the
pure
gel
hydrogel has the enzyme-responsive function of α-amylase.
performance of α-amylase-responsive gel was recorded. When the α-amylase was added into the
hydrogel after 10 min at 37 °C, the hydrogel collapsed. The result is shown in the optical images of
Figure 4b. This result is due to the α-CD in the gel hydrolyzed by α-amylase. This means that the
hydrogel has the enzyme-responsive function of α-amylase.
Figure 4. (a) The fluorescence spectrum of the hydrogel and two-component solution at
Figure 4. (a) The fluorescence spectrum
of the hydrogel and two-component solution at room
room temperature ([NPPD] = 1.0 × 10−6 mol/L, [RF] = 2 × 10−6 mol/L, [α-CD] = 1.0 × 10−6 mol/L,
−6 mol/L, [RF] = 2 × 10−6 mol/L, [α-CD] = 1.0 × 10−6 mol/L,
temperature
([NPPD]
=
1.0
×
10
[α-amylase] = 0.001 mol/L). (b) The optical images show the gel hydrolyzed by α-amylase.
[α-amylase] = 0.001 mol/L). (b) The optical images show the gel hydrolyzed by α-amylase.
Figure 4. (a) The fluorescence spectrum of the hydrogel and two-component solution at
As further evidence of the enzyme responsiveness
of the gel, the surface morphology after the
room temperature ([NPPD] = 1.0 × 10−6 mol/L, [RF] = 2 × 10−6 mol/L, [α-CD] = 1.0 × 10−6 mol/L,
gel was hydrolyzed was observed by a scanning electron microscope, as shown in Figure 5. In the
[α-amylase]
= 0.001
(b) The optical
images show theof
gelthe
hydrolyzed
by surface
α-amylase.morphology after the
As further
evidence
ofmol/L).
the enzyme
responsiveness
gel, the
figure, the close-knit structure was observed, in which fine-spun fibers with a diameter ranging
gel wasfrom
hydrolyzed
observed by
a scanning
microscope, as shown
in after
Figure
0.1
0.7 was
um
intertwined
with
each
otherelectron
to form
networks
the5. In the
As to
further
evidence
of the enzyme
responsiveness
of thethree-dimensional
gel, the surface morphology
after the
figure, xerogel
the
close-knit
structure
was
observed,
in
which
fine-spun
fibers
with
a
diameter
ranging
from
hydrolyzed
by α-amylase
(Figure electron
5a,b). The
fluorescence
of the
hydrogel
gel waswas
hydrolyzed
was observed
by a scanning
microscope,
as shown
in Figure
5. In after
the
0.1 to 0.7
um intertwined
each
other
to
form
three-dimensional
after
theupon
xerogel was
α-amylase
processing
could
be observed
under
a confocal
laser scanning
microscope
(CLSM)
figure,
the
close-knitwith
structure
was
observed,
in
which fine-spun
fibersnetworks
with a diameter
ranging
excitation
atto405
was observed
that
the
compact
network
were
composed
of long
fromby
0.1α-amylase
0.7nm.
umIt(Figure
intertwined
with
each
other
to form
networks
after
the
hydrolyzed
5a,b).
The
fluorescence
ofthree-dimensional
thestructures
hydrogel
after
α-amylase
processing
and
interlaced
fibers
without
(Figure
When
the (CLSM)
α-CD was
hydrolyzed,
theafter
π–π
xerogel
was under
hydrolyzed
byfluorescence
α-amylase
(Figure S6c,d).
5a,b).
The fluorescence
ofupon
the hydrogel
could be
observed
a confocal
laser scanning
microscope
excitation
at 405 nm.
stacking
between
NPPD
andbe
RF/NPPD
stronger.
Then,laser
thescanning
fluorescence
was quenching.
The
α-amylase
processing
could
observed is
under
a confocal
microscope
(CLSM) upon
It was observed that the compact network structures were composed of long and interlaced fibers
excitation at
nm.
It was observed
that the compact
network
structures
were
of long
morphology
of405
these
structures
upon excitation
at 405 nm
was consistent
with
thecomposed
SEM images.
without fluorescence
S6c,d).
When the
α-CDS6c,d).
was hydrolyzed,
the
π–π
stackingthe
between
NPPD
and interlaced (Figure
fibers without
fluorescence
(Figure
When the α-CD
was
hydrolyzed,
π–π
and RF/NPPD
is
stronger.
Then,
the
fluorescence
was
quenching.
The
morphology
of
these
structures
stacking between NPPD and RF/NPPD is stronger. Then, the fluorescence was quenching. The
morphology
of these
upon excitation
at 405
nmimages.
was consistent with the SEM images.
upon excitation
at 405
nm structures
was consistent
with the
SEM
Figure 5. The SEM images of the gel after α-amylase processing ([NPPD] = 10−3 mol/L,
[α-CD] = 10−3 mol/L, [RF] = 2 × 10−3 mol/L, [α-amylase] = 0.01 U·mL−1), (a) the xerogels after
α-amylase processing in a mixed solution for a larger scale; (b) For hydrogel in a mixed solution
in a
FigureThe
5. The
images ofof the
the gel
processing
([NPPD]([NPPD]
= 10−3 mol/L,
Figure 5.
SEMSEM
images
gel after
afterα-amylase
α-amylase
processing
= 10−3 mol/L,
small
scale.
[α-CD] = 10−3 mol/L, [RF] = 2 × 10−3 mol/L, [α-amylase] = 0.01 U·mL−1), (a) the xerogels after
[α-CD] = 10−3 mol/L, [RF] = 2 × 10−3 mol/L, [α-amylase] = 0.01 U·mL−1 ), (a) the xerogels after
α-amylase processing in a mixed solution for a larger scale; (b) For hydrogel in a mixed solution in a
α-amylase
processing in a mixed solution for a larger scale; (b) For hydrogel in a mixed solution in a
small scale.
small scale.
Chemosensors 2017, 5, 6
6 of 8
4. Conclusions
We have discovered an enzyme-responsive three-component hydrogel, which was composed
of amino acids-functionalized perylene derivatives, riboflavin and α-CD. The hydrogels exhibited
a specific α-amylase response and fluorescence properties. Such properties open the possibility for
hydrogels to become potential materials in the field of drug delivery and stimulation-response.
Supplementary Materials: The following are available online at www.mdpi.com/2227-9040/5/1/6/s1.
Acknowledgments: The research was supported by National Natural Science Foundation of China (No. 21365003,
21463003, 51478123). The Jiangxi outstanding youth fund (20153BCB23001) and Jiangxi Provincal project of
scientific and technological innovation team (20152BCB24008) are also gratefully acknowledged.
Author Contributions: Y.L. conceived and designed the experiments; W.L. and Y.F. performed the experiments;
Y.P. and Y.W. analyzed the data; X.L. and X.F. contributed reagents/materials/analysis tools; Y.P. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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