A colorimetric method for α-glucosidase activity assay … · reversibly bind diols with high...

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

mailto:[email protected]

Nano Res

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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:

[email protected].







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

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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:


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


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


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


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


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


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


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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