a colorimetric method for α-glucosidase activity assay … · reversibly bind diols with high...
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Nano Res
1
A colorimetric method for α-glucosidase activity assay
and its inhibitor screening based on aggregation of
gold nanoparticles induced by specific recognition
between phenylenediboronic acid and 4-aminophenyl-
α-D-glucopyranoside
Juan Zhang1, Ying Liu1, Jun Lv1, Genxi Li1,2 ()
Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-014-0573-1
http://www.thenanoresearch.com on Aughst 27, 2014
© Tsinghua University Press 2014
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() Corresponding author at: Department of Biochemistry, Nanjing University, P. R. China, Fax: +86 25 83592510, E-mail address: [email protected].
Nano Research
DOI 10.1007/s12274-014-0573-1
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A colorimetric method for α-glucosidase activity assay
and its inhibitor screening based on aggregation of
gold nanoparticles induced by specific recognition
between phenylenediboronic acid and 4-aminophenyl-
α-D-glucopyranoside
Juan Zhang1, Ying Liu1, Jun Lv1, Genxi Li1,2
1 Laboratory of Biosensing Technology, School of Life
Sciences, Shanghai University, Shanghai 200444, P. R.
China
2 State Key Laboratory of Pharmaceutical Biotechnology,
Department of Biochemistry, Nanjing University, Nanjing
210093, P. R. China
New colorimetric method for α-glucosidase activity assay and
its inhibitor screening based on aggregation of gold nanoparti-
cles induced by specific recognition between phenylenedibronic
acid and 4-aminophenyl-α-D-glucopyranoside.
Corresponding author at: Department of Biochemistry, Nanjing University, P. R. China, Fax: +86 25 83592510, E-mail address:
A colorimetric method for α-glucosidase activity assay
and its inhibitor screening based on aggregation of
gold nanoparticles induced by specific recognition
between phenylenediboronic acid and 4-aminophenyl-
α-D-glucopyranoside
Juan Zhang1, Ying Liu1, Jun Lv1, Genxi Li1,2 ()
Received: day month year
Revised: day month year
Accepted: day month year
(automatically inserted by
the publisher)
© Tsinghua University Press
and Springer-Verlag Berlin
Heidelberg 2014
KEYWORDS
α-glucosidase,
inhibitor screening,
1,4-phenylenediboronic
acid,
gold nanoparticles
ABSTRACT
A colorimetric method has been established for α-glucosidase activity assay and
its inhibitor screening in this work. The method is based on the specific
recognition between 1,4-phenylenediboronic acid (PDBA) and 4-aminophenyl-
α-D-glucopyranoside (pAPG), which may induce aggregation of pAPG
functionalized gold nanoparticles (AuNPs) to achieve color change of the test
solution. Because pAPG is the substrate of α-glucosidase, the aggregation of
AuNPs will be influenced by α-glucosidase since there is no coordination
reactivity between PDBA and 4-aminobenzene, the hydrolyzed product of
pAPG catalyzed by the enzyme. Therefore, a simple and easily-operated
colorimetric method for the assay of α-glucosidase activity can be developed.
Under the optimized experimental conditions, the ratios of absorbance at the
wavelength of 650 nm to that at 520 nm are linear with the α-glucosidase
activity within a range from 0.05 to 1.1 U/mL with a lowest detection limit of
0.004 U/mL. Moreover, using the proposed method, the inhibition effect of
gallic acid and quercetin on α-glucosidase activity can be tested with IC50 values
of 1.16 mM and 1.82 μM, respectively. Thus, the method has a great potenial not
only for the detection of α-glucosidase activity but also the screening of its
inhibitor.
() Corresponding author at: Department of Biochemistry, Nanjing University, P. R. China, Fax: +86 25 83592510, E-mail address: [email protected].
Nano Research
DOI (automatically inserted by the publisher)
Research Article
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2 Nano Res.
1 Introduction
Type II diabetes mellitus (T2DM) is a serious
metabolic disorder, which is diagnosed on the basis
of sustained hyperglycemia [1]. Intensive control on
blood glucose level is always an effective way to halt
T2DM development [2]. It is well-known that
α-glucosidase is a membrane-bound enzyme located
at the epithelium of small intestine, and the key
enzyme of carbohydrate digestion [3]. It specifically
hydrolyzes the α-glucopyranoside bond, thereby
releasing α-D-glucose from the non-reducing end of
sugar [4]. Inhibitors of α-glucosidase would delay the
digestion and absorption of carbohydrates and
suppress the postprandial hyperglycemia [5, 6].
Therefore, it is of great importance to develop
sensitive and easy-to-use methods to assay the
enzyme so as to screen new and more effective
α-glucosidase inhibitors.
Currently, hyperglycemic animal model [7] for in
vivo screening and enzyme-inhibitor model [8] for in
vitro screening have been routinely employed for
α-glucosidase inhibitor screening. The hyperglycemic
animal model can provide relatively reliable results,
but it involves expensive and long-term animal
experiments. The enzyme-inhibitor model, based on
para-nitrophenyl-α-D-glucopyranoside (pNPG) sub-
strate, has been widely used to screen active
inhibitors from plants, but its sensitivity is often
limited to some extent because the activity of
α-glucosidase inhibitors is usually quantified by
measuring absorbance of 4-nitrophenol at 400 nm
released from pNPG [9]. Another drawback is that
the detection will be disturbed when the absorption
of inhibitor overlaps with that of 4-nitrophenol [10].
Benefiting from the unique optical properties of
gold nanoparticles (AuNPs), increasing attention has
been attracted by AuNP-based colorimetric assays
owing to the designability of the surface chemistry of
AuNPs and the technical simplicity, high sensitivity
[11] and selectivity of the methods [12]. Up to now,
AuNPs-based colorimetric methods have been
widely used for testing different kinds of species like
nucleic acids [13-16], proteins [17-19], metal ions [20,
21], and so on.
Boronic acid is an excellent molecular receptor for
saccharides [22]. Boronic acids are also known to
reversibly bind diols with high affinity to form cyclic
esters [23]. Herein, based on these findings, a
colorimetric method has been proposed in this work
for α-glucosidase activity assay and its inhibitor
screening. This method is based on specific
recognition between phenylenediboronic acid and
glycogen part of the enzyme substrate that is linked
to AuNPs. Consequently, aggregation of the
functionalized AuNPs can be triggered to cause the
color change of the reaction system. To the best of
our knowledge, this is the first report for
α-glucosidase activity assay and the inhibitor
screening with a simple AuNP-based colorimetric
method. Furthermore, since this method can be used
in a high-throughput fashion by the naked eye or a
simple colorimetric reader, it may have great
potential applications in the future.
2 Experimental section
2.1 Materials and Reagents
Chloroauric acid (HAuCl4·3H2O), trisodium citrate,
3-mercaptopropionic acid (MPA), and α-glucosidase
(EC 3.2.1.20, from Saccharomyces cerevisiae) were
purchased from Sigma (Shanghai, China).
1,4-Phenylenediboronic acid (PDBA) was obtained
from Aladdin (Shanghai, China). 4-Aminophenyl-α-
D-glucopyranoside (pAPG) was purchased from
Beijing Chemsynlab Pharmaceutical Co. Ltd (Beijing,
China). All buffers and aqueous solutions were
prepared with ultrapure water, which was purified
with a Millipore Milli-Q water purification system
(Branstead, USA) to a specific resistance of 18 MΩ
cm.
The solution of α-glucosidase was obtained by
dissolving at different concentration in 10 mM PBS
buffer (pH 6.8). PDBA stock solution was prepared
by dissolving at a concentration of 300 mM in 95%
ethanol and then diluting with 10 mM PBS buffer
(pH 6.8). In the reaction solution, the highest
resulting ethanol concentration was about 1%, so
there was no appreciable effect on protein structure.
2.2 Synthesis of the functionalized gold
nanoparticles
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3 Nano Res.
AuNPs were synthesized according to a published
procedure [24]. In brief, trisodium citrate (10 mL, 38.8
mM) was added to a boiling solution of HAuCl4 (100
mL, 1 mM), and the resulting solution was kept
continuously boiling for another 30 min to give a
wine red mixture. The mixture was cooled to room
temperature and then filtrated through a Millipore
syringe (0.45 μm) to remove the precipitate, and the
filtrate was stored in a refrigerator at 4 °C for use.
MPA was covalently attached to AuNPs according
to a previous report [25]. Briefly, MPA was dissolved
in anhydrous ethanol to obtain its concentration of 1
mM, followed by the adjustment of the pH value to
11. Then, 1 mL MPA solution was added into 40 mL
of AuNP solution. At last, the mixed solution was
stirred at room temperature for 3 h to give the
middle product, MPA/AuNPs.
The enzyme substrate, pAPG, was further
covalently linked to MPA/AuNPs. 1 mL 1 mM pAPG
aqueous solution was firstly added to 40 mL
MPA/AuNPs solution. Then the mixed solution was
stirred at room temperature for 12 h to give the final
product, pAPG/MPA/AuNPs.
2.3 Characterization of the functionalized gold
nanoparticles
For the characterization of the functionalized AuNPs,
transmission electron microscope (TEM) (JEOL,
Japan) was firstly used to detect the shape and size.
For TEM measurements, samples were prepared by
placing drop of sample solution on a carbon-coated
standard copper grid (300 meshes) operating at 80 kV.
Meanwhile, Fourier transforms infrared spectroscopy
(FT-IR) was used to detect the functionalized AuNPs.
FT-IR spectra were obtained on a VERTEX70 Fourier
Transform Infrared spectrometer (Bruker Co. Ltd.,
Bergisch Gladbach, Germany) fitted with DTGS
(deuterated triglycine sulphate) detector. The spectra
were recorded in transmission mode from 4000 to
400 cm-1 at a resolution of 0.44 cm-1 at room
temperature.
Moreover, the hydrodynamic sizes and zeta
potentials of the synthetic nanoparticles was further
confirmed by dynamic light scattering (DLS) with a
commercial Zeta sizer (Malvern Instruments, UK).
The samples were first loaded into disposable cells
and the particle sizes were tested in triplicate. For
zeta potential measurements, samples were loaded in
regular disposable cells and measurements were
performed using a dip probe.
2.4 α-Glucosidase activity assay
Firstly, the solution of PDBA (30 μL) was added into
a dispersion of pAPG/MPA/AuNPs (60 μL). Then,
the resulting mixture was incubated at room
temperature for 30 min. After that, photographs were
taken with a digital camera (NET-3NL, SONY, Japan)
and UV-vis spectra were recorded with a
spectroscopic instrument (Shimadzu Co., Kyoto,
Japan). The final concentrations of PDBA in the
resulting mixtures were 1.50, 1.65, 1.80, 1.95, 2.10,
2.25, 2.40, 2.55, 2.70, 2.85, and 3.00 mM.
α-Glucosidase activity assay was performed under
the following procedures. The enzyme solution was
firstly obtained through the dissolution of
α-glucosidase into phosphate buffer solution (10 mM,
pH 6.8). After that, 10 µL of enzyme solution with
different concentrations ranging from 0.05 to 1.1
U/mL was added to 60 µL of dispersed
pAPG/MPA/AuNPs solution, and the resulting
mixture was allowed to stand by at 37°C for 20 min.
Then, 30 µL PDBA was added into the resulting
mixture at room temperature. After 15 min, the
reaction solution were photographed and used for
UV-vis spectroscopic measurements.
2.5 Inhibition efficiency evaluation
In the inhibition assay, gallic acid or quercetin with
different concentrations (10 µL) were firstly
premixed with α-glucosidase (10 µL, 1.1 U/mL) for
10 min at 37°C. Then pAPG/MPA/AuNPs (60 µL)
were added into the mixed solution and
continuously incubated for 20 min at 37°C. After that,
PDBA (30 µL) was added into the resulting mixture
to obtain its final concentration of 3.00 mM. At last,
the reaction solution was allowed to stand by at room
temperature for 15 min. The absorbance of each
sample was recorded using UV-vis spectroscopy with
the IC50 value calculated. The inhibitory ratio (%) of
gallic acid or quercetin on α-glucosidase was
expressed as follows:
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4 Nano Res.
0 0
650 520 650 520
0 0
650 520
/ - /Inhibitory ratio (%) = 100
/
A A A A
A A
where A650/A520 was the ratio of the absorbance value
at 650 nm to that at 520 nm in the presence of both
the inhibitor and the enzyme, A0
650/A0
520 was the ratio
of the absorbance value at 650 nm to that at 520 nm in
the presence of the enzyme.
3 Results and discussion
The principle of the colorimetric method is shown in
Scheme 1. Firstly, MPA and pAPG are in turn linked
to AuNPs to form the dispersed functionalized
AuNPs (pAPG/MPA/AuNPs) through covalent Au-S
and amide bonds, respectively (Scheme 1(a)).
Subsequently, the addition of 1,4-phenylenedibronic
acid (PDBA) as a linker can trigger the aggregation of
AuNPs due to the high coordination reactivity
between PDBA and pAPG (Scheme 1(c)). However, in
the presence of α-glucosidase, the aggregation will
not happen resulted from the specific cleavage of
pAPG into 4-aminobenzene which has no binding
ability with PDBA (Scheme 1(b)). Since the
concentration of the enzyme is related to the
aggregated degree of AuNPs, a new method for
monitoring α-glucosidase activity through the
change in the UV-vis spectrum of the AuNP
dispersion, or even through the direct observation by
naked eye, can be developed. Furthermore, this
method can also be used to evaluate the inhibitor of
α-glucosidase (Scheme 1(d)).
3.1 Synthesis and characterization of pAPG/MPA/
AuNPs
As illustrated in Scheme 1(a), the functionalized
AuNPs are prepared through two steps. Firstly, MPA
is linked to AuNPs to form MPA-modified AuNPs
(MPA/AuNPs), based on the efficient displacement of
citrate with MPA by adjusting the solution to pH 11
with NaOH. Secondly, pAPG is conjugated to the
MPA/AuNPs surface through chemical reaction
between the amine groups of pAPG and the
carboxylic acid groups of MPA to give the
functionalized AuNPs (pAPG/MPA/AuNPs). As
shown in Fig. 1(d), compared pAPG/MPA/AuNPs
with pAPG, two new peaks located at 1654 and 1538
cm-1, which belong to stretching vibration of C=O
(amide I) and bending vibration of N-H (amide II),
respectively, may demonstrate the formation of
amide bond between pAPG and MPA/AuNPs.
Fig. 1(a) and 1(b) separately shows the TEM image
of AuNPs and pAPG/MPA/AuNPs. The mean
diameters of AuNPs and pAPG/MPA/AuNPs are
14.84 ± 0.39 and 16.65 ± 0.09 nm, respectively. So, after
Scheme 1 Schematic illustration of the mechanisms for (a) the synthesis of functionalized AuNPs, (b) the catalytic reaction of
α-glucosidase, (c) specific recognization between phenylenediboronic acid and glycogen, (d) colorimetric determination of
α-glucosidase activity and the inhibitor
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5 Nano Res.
Figure 1 TEM images of (a) the citrate-stabilized AuNPs, (b) the functionalized AuNPs (pAPG/MPA/AuNPs) and (c) aggregation of
pAPG functionalized AuNPs triggered by PDBA. The size distribution histograms of the citrate-stabilized AuNPs (a, inset) and the
functionalized AuNPs (b, inset) were made from the TEM images by counting more than 100 particles. (d) The FT-IR spectra of pAPG
and pAPG/MPA/AuNPs. (e) The zeta potential (ζ) of AuNPs, MPA/AuNPs, and pAPG/MPA/AuNPs (pH=7.0). (f) UV-vis spectra of
AuNPs, MPA/AuNPs, and pAPG/MPA/AuNPs.
functionalization, the nanoparticles show an
increased diameter (1.81 nm) and it suggests that the
layer of both MPA and pAPG has an approximate
size of 1-2 nm.
It has been reported that DLS is useful for
determining the zeta potential (ζ) of nanoparticles in
Mill-Q H2O [26]. The surface charge of AuNPs and
the pH of the solution directly relate to the stability
of AuNPs. The zeta potential of all three types of
AuNPs has been measured at pH 6.8 to verify the
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6 Nano Res.
stability. Zeta potentials of AuNPs, MPA/AuNPs, and
pAPG/MPA/AuNPs are -35.70 ± 3.75, -21.24 ± 6.11,
and -2.78 ± 2.37 mV, respectively (Fig. 1(e)). When
AuNPs have been modified successively with MPA
and pAPG, the values of zeta potential become
decreasingly negative, which also clearly indicates
that MPA/AuNPs and pAPG/MPA/AuNPs conju-
gates can be formed, and they both are stable.
Moreover, the surface plasmon bands of AuNPs,
MPA/AuNPs, and pAPG/MPA/AuNPs remain
unchanged (Fig. 1(f)).
3.2 Mechanism investigation for enzyme activity
assay
As shown in Fig. 2, addition of PBS buffer to the
pAPG functionalized AuNP solution as a control
experiment, the red color of the test solution keeps
unchanged, and the solution exhibits a strong
absorption at 520 nm (vial 1, black curve), showing
the feature of well-dispersed AuNPs [27].The
addition of α-glucosidase (vial 2, red curve) into the
AuNP solution does not result in an observable
aggregation of AuNPs either, thus the test solution
keeps the initial color and UV−vis spectrum of well
dispersed AuNPs. These results suggest that the sole
addition of α-glucosidase does not induce the
aggregation of AuNPs. Nevertheless, it should be
noticed from Fig. 2 that addition of PDBA, the linker
molecule, may essentially trigger the aggregation of
AuNPs, resulting in the change of the color from
initially wine red to blue and the production of a new
absorption peak at 650 nm (vial 3, blue curve).
Typically, a change in the color of the AuNP solution
is caused by particle aggregation (or agglomeration)
and light scattering-assisted surface plasmon, which
has been confirmed by TEM. As shown in Fig. 1(c),
the addition of PDBA leads to the aggregation of the
pAPG functionalized AuNPs. This phenomenon can
be explained by the specific molecular recognization
between PDBA and saccharide fraction of pAPG on
the surface of AuNPs. Both 2'-OH and 4'-OH in
saccharide fraction of pAPG can coordinate with
boric acid group of PDBA, forming a stable
six-membered ring of the complex (Scheme 1(c)) [28],
so the surface coordination interaction may cause the
aggregation of AuNPs, being accountable for the
Figure 2 UV-vis spectra and photographs (inset) of the mixtures
prepared by separate addition of PBS buffer (40 μL, 10 mM, pH
6.8) (vial 1, black curve), α-glucosidase (10 μL, 1.1 U/mL) (vial
2, red curve), PDBA (30 μL, 8.5 mM) (vial 3, blue curve), or
α-glucosidase (10 μL, 1.1 U/mL) + PDBA (30 μL, 8.5 mM) (vial
4, cyan curve), into pAPG/MPA/AuNPs (60 μL).
alternation of both the color and the UV-vis spectrum
of the aqueous dispersion of the AuNPs. On the other
hand, it should be noticed from Fig. 2 that the
successive addition of α-glucosidase and PDBA into
the aqueous solution of the pAPG/MPA/AuNPs, both
the color and the UV-vis spectra of the solution
maintain almost unchanged (vial 4, cyan curves), so
occurrence of aggregation of AuNPs is hardly
achieved. This can be attributed to the remove of
saccharide fraction from the surface of AuNPs to the
solution, owing to the cleavage of glycoside bond
hydrolyzed by α-glucosidase (Scheme 1(b)), thus
PDBA cannot link the nanoparticles to result in their
aggregation.
The mechanism of the specific recognition between
glycogen and PDBA is displayed in Fig. 3(a). PDBA
acts as week Lewis acid, and it is able to react with
water to transfer from the neutral trigonal form to the
anionic tetrahedral form [29]. In the presence of
pAPG/MPA/AuNPs, a six-membered cyclic
phenylenediboronate monoester (I) will form
through the interaction with both 2'-OH and 4'-OH of
glycogen group at the out layer of AuNPs.
Subsequently, two six-membered cyclic phenylenedi-
boronate diester further produces (II), causing the
aggregation of AuNPs. In the aqueous solution, the
phenylenediboronate monoester (I) is in equilibrium
with the phenylenediboronate diester (II). Hence, the
concentration of PDBA in the reaction
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7 Nano Res.
Figure 3 (a) Illustration for the mechanism of the specific recognition between glycogen and phenylenediboronic acid. (b) Kinetic plots
of time-dependent A650/A522 values versus those with different concentrations of PDBA. Digital photographs at a time point of 30 min
after the addition of different concentrations of PDBA are shown on the right. Right, from vial 1 to vial 12: the concentrations of PDBA
were 0, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 mM. (c) Initial rate of coordination reaction (v0) versus PDBA concentration. Error
bars indicate standard deviations (n = 3).
solution will have an evident influence on the
sensitivity of the detection, and the effect of PDBA
concentration on the detection system has been
further investigated. The UV-Vis spectra of the
reaction solution have been successively recorded for
30 min after the addition of PDBA with different
concentrations (Fig. S1). It has been demonstrated
that the value of A650 to A520 (A650/A520) could be
considered as an indicator for the degree of
dispersion/aggregation state of AuNPs [30-32]. As
exhibited in Fig. 3(b), the time-dependent A650/A520
values from the UV-vis spectra of AuNPs has been
summarized in the presence of different
concentrations of PDBA in order to estimate the
kinetics of the coordination between pAPG and
PDBA. The A650/A520 values increase with the
increasing concentrations of PDBA. Meanwhile, the
color of the aqueous dispersions of the AuNPs
gradually changes from wine red to violet blue (right,
from vial 1 to vial 12) at the time point of 30 min
when the concentrations of PDBA increase from 0 to
10 mM. These results signify the kinetics of AuNP
aggregation becomes faster with the increasing
PDBA concentrations in the dispersions. The initial
reaction rate (v0) can be obtained by calculating the
slopes of the initial part of the kinetic curves of
different PDBA concentrations (Fig. 3(c)). With the
increase of PDBA concentration from 5 to 10 mM, the
initial reaction rate gradually raises. When the
concentration of PDBA attains to 8.5 mM, the
changes of the rate decrease. Furthermore, the
ultimately A650/A520 values almost keep unchanged
when the concentration of PDBA varies from 8.5 to 10
mM (Fig. 3(b)). Therefore, 8.5 mM is chosen as the
optimal reaction concentration for this study.
3.3 α-Glucosidase activity assay
As shown in Fig. 4, with the increase of the
α-glucosidase concentration from 0 to 1.1 U/mL, the
color of the dispersion gradually turns from violet to
wine red (from vial 1 to vial 8), and a dramatic
change occurs in the UV-vis spectra of the dispersion.
Meanwhile, with the increasing concentration of
α-glucosidase, A650 decreases, while A520 increases.
Owing to the breakage of α-glucosidic bond in the
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8 Nano Res.
Figure 4 UV-vis spectra and photographs (above) of the mixtures
prepared by the addition of various concentrations of
α-glucosidase (10 μL) into the dispersion of pAPG/MPA/AuNPs
followed by the addition of PDBA (30 μL, 8.5 mM). Above, from
vial 1 to vial 8: the α-glucosidase concentrations were 0, 0.05, 0.1,
0.3, 0.5, 0.7, 0.9, and 1.1 U/mL. Inset: Plot of A650/A520 against
the α-glucosidase concentration. Error bars indicate standard
deviations (n = 3).
presence of α-glucosidase, the saccharide fraction
located at the out layer of the AuNPs will depart
from the surface of AuNPs and be converted into
α-glucose dissolving in the aqueous solution, which
will lead to the declined extent of the aggregation of
the AuNPs. The ratio of the absorbance value at 650
nm to that at 520 nm (A650/A520) has been used for the
quantitative detection of the α-glucosidase activity.
As depicted in Fig. 4 (Inset), the values of A650/A520
linearly decrease with the increase of α-glucosidase
concentrations, corresponding to the lessened AuNP
aggregation and the reduced color change. The ratio
of A650/A520 shows a linear response toward
α-glucosidase within a concentration range from 0.05
to 1.1 U/mL, which is wider than the previous report
[33], and follows the regression equation of A650/A520 =
1.358 - 1.112 c (U/mL, R = 0.989). Furthermore, the
detection limit has also been calculated to be 0.004
U/mL by the interpolation of the mean plus three
times the standard deviation of the zero standards.
The detection precision has been investigated
according to the slope of the regression of
α-glucosidase (from 0.05 to 1.1 U/mL) obtained from
three independent assay processes. The RSD of the
three slopes is 1.81%, suggesting that the proposed
method has good precision.
3.4 Inhibition assay for enzymatic activity
It has been reported that specific α-glucosidase
inhibition may decreases postprandial
hyperglycemia and improves impaired glucose
tolerance. As a kind of oral anti-diabetic agents,
α-glucosidase inhibitor can be able to avoid safety
and tolerability issues of conventional agents. In
order to test and verify that the developed method
can be employed for the screening of α-glucosidase
inhibitors, two compounds, gallic acid and quercetin,
have been selected used for this study.
As exhibited in Fig. 5(a), with increasing gallic acid
concentrations from 0.4 to 1.2 mM, the color of the
dispersion changes from wine red to purple (inset,
from vial 1 to vial 9), corresponding to the decrease
in A520 and the increase in A650. The value of A650/A520
first increases gradually with the increasing
concentration and then trends to level off when the
inhibition concentrations achieve a certain level (Fig.
S2(a)). This dedicates that the inhibition of gallic acid
is in a dose-dependent manner. It has been reported
that the gallic acid can competitively inhibit
α-glucosidase [34]. Gallic acid and pAPG/MPA/
AuNPs can competitively bind the active site of
α-glucosidase so as to prevent the cleavage of
glycosidic bond by the enzyme and promote the
aggregation of AuNPs. It is found that 1.1 mM gallic
acid can remarkably inhibit the activity of 0.1 U/mL
α-glucosidase because there is no evident change in
either UV-vis spectra or the color of dispersion (Fig. 5
(a), vial 8). The maximum inhibition of gallic acid is
48.98% with the IC50 value of 1.16 mM (Fig. 5(b)),
which is in accordance with the values reported
previously [35, 36].
Different from that of gallic acid, a competitive
inhibitor, the inhibitory mode of quercetin toward
α-glucosidase is a mixture type of noncompetitive
and anticompetitive [37, 38], so quercetin has been
further used to evaluate the proposed method.
Same as gallic acid, along with the increase of
quercetin concentrations from 0.2 to 12.6 μM, the
color of the dispersion gradually turns from wine
red to purple (inset, from vial 1 to vial 9), in
consistent with the decrease in A520 and the increase
in A650 (Fig. 5(c)). As shown in Fig. S2(b), the ratio of
A650/A520 first increases steadily with the increasing
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9 Nano Res.
Figure 5 UV-vis spectra and photographs (inset) of the dispersion upon analyzing various concentrations of (a) gallic acid and (c)
quercetin. Inset (a), vial 1 to vial 9: the gallic acid concentrations were 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, and 1.2 mM. Inset (c), vial
1 to vial 9: the quercetin concentrations were 0.2, 0.9, 1.8, 3.6, 5.4, 7.2, 9, 10.8, and 12.6 µM. Inhibition ratio versus (b) the gallic
acid and (d) quercetin concentration. Error bars indicate standard deviations (n = 3).
concentration. Then, the ratio remains unchanged
when the quercetin concentration attains to 10.8 µM.
It implies that the inhibition of quercetin is a
dose-independent mode. The inhibition effect of
quercetin on α-glucosidase can be attributed to the
unsaturated C ring, 3-OH, 4-CO, and the hydroxyl
substitution on the B ring [39]. Moreover, the 3'-OH
and 4'-OH of quercetin can interact with Asp 214
and Glu 276 of α-glucosidase active site amino acid
by hydrogen bonds, thus preventing the cleavage of
glycoside bond on the surface of pAPG/MPA/
AuNPs and stimulate the aggregation of AuNPs. It
is observed that the activity of 0.1 U α-glucosidase
can be significantly inhibited with the addition of
10.8 µM quercetin, since no obvious change in
either UV-vis spectra or the color of the dispersion
can be investigated (Fig. 5(c), vial 8). The maximum
inhibition ratio of quercetin is 76.48% with the IC50
value of 1.82 µM (Fig. 5(d)), in consistent with the
previous report [40]. These results essentially
demonstrate that the colorimetric method could
also be used to evaluate the inhibitor efficiency.
4 Conclusions
In summary, a simple and yet effective colorimetric
method has been firstly demonstrated for
α-glucosidase activity assay and the inhibitor
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10 Nano Res.
screening based on specific recognition between
pAPG and PDBA. The method bears advantages in
the low technical and instrumental demands, wide
detection range, and high sensitivity, and thus it will
have a great potential for the detection of
α-glucosidase activity and the screening of the
inhibitor.
Acknowledgements
This work is supported by the National Natural
Science Foundation of China (Grant Nos. 31101354
and 21235003).
Electronic Supplementary Material: Supplementary
material is available in the online version of this
article at http://dx.doi.org/10.1007/s12274-***-****-*
(automatically inserted by the publisher).
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