Study of the inhibition of two human maltase-glucoamylases catalytic domains by different α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.carres.2011.09.012

In humans, both the N-terminal catalytic domain (NtMGAM) and the C-terminal catalytic domain (CtMGAM) of small intestinal maltase glucoamylase (MGAM) are α-glycosidases that catalyze the hydrolysis of α-(1→4) glycosidic linkages in the process of starch digestion, and are considered to be the main therapeutic targets for type 2 diabetes. In this work, recombinant human CtMGAM has been cloned for the first time, and this, combined with the expression of NtMGAM in Pichia pastoris, made it possible for us to study the catalytic mechanism of MGAM in a well-defined system. The enzymatic kinetic assays of the two catalytic domains suggest that CtMGAM has the higher affinity for longer maltose oligosaccharides. Kinetic studies of commercially-available drugs such as 1-deoxynojirimycin (DNJ), miglitol, voglibose, and acarbose along with a series of acarviosine-containing oligosaccharides we isolated from Streptomyces coelicoflavus against NtMGAM, CtMGAM, and human pancreatic α-amylase (HPA) provide us an overall profile of the inhibitory ability of these inhibitors. Of all the inhibitors used in this paper, DNJ was the most effective inhibitor against MGAM; the K_i values for the two catalytic domains were 1.41 and 2.04 μM for NtMGAM and CtMGAM, respectively. Acarviostatins 2-03 and 3-03 were the best inhibitors against HPA with relatively high inhibitory activity against CtMGAM. The acarviostatins 2-03 and 3-03 inhibition constants, K_i, for HPA were 15 and 14.3 nM, and those for CtMGAM were 6.02 and 6.08 μM, respectively. These results suggest that NtMGAM and CtMGAM differ in their substrate specificities and inhibitor tolerance despite their structural relationship.

Full text of DOI:10.1016/j.carres.2011.09.012

Carbohydrate Research 346 (2011) 2688–2692
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Study of the inhibition of two human maltase-glucoamylases catalytic
domains by different
a-glucosidase inhibitors
Limei Ren a, Xiaofang Cao a, Peng Geng b, Fang Bai a, , Gang Bai a
⇑
a College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China
b Basic Medical College, Tianjin Medical University, Tianjin 300070, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In humans, both the N-terminal catalytic domain (NtMGAM) and the C-terminal catalytic domain (CtM-
Received 9 August 2011
Received in revised form 15 September
2011
Accepted 17 September 2011
Available online 24 September 2011
GAM) of small intestinal maltase glucoamylase (MGAM) are
a-glycosidases that catalyze the hydrolysis
of -(1?4) glycosidic linkages in the process of starch digestion, and are considered to be the main ther-
a
apeutic targets for type 2 diabetes. In this work, recombinant human CtMGAM has been cloned for the
first time, and this, combined with the expression of NtMGAM in Pichia pastoris, made it possible for
us to study the catalytic mechanism of MGAM in a well-defined system. The enzymatic kinetic assays
of the two catalytic domains suggest that CtMGAM has the higher affinity for longer maltose oligosaccha-
rides. Kinetic studies of commercially-available drugs such as 1-deoxynojirimycin (DNJ), miglitol, vogli-
bose, and acarbose along with a series of acarviosine-containing oligosaccharides we isolated from
Keywords:
Maltase-glucoamylase
Human pancreatic a-amylase
1-Deoxynojirimycin
Streptomyces coelicoflavus against NtMGAM, CtMGAM, and human pancreatic
a-amylase (HPA) provide
us an overall profile of the inhibitory ability of these inhibitors. Of all the inhibitors used in this paper,
Acarviostatins
DNJ was the most effective inhibitor against MGAM; the Ki values for the two catalytic domains were
1.41 and 2.04 lM for NtMGAM and CtMGAM, respectively. Acarviostatins 2-03 and 3-03 were the best
inhibitors against HPA with relatively high inhibitory activity against CtMGAM. The acarviostatins 2-
03 and 3-03 inhibition constants, Ki, for HPA were 15 and 14.3 nM, and those for CtMGAM were 6.02
and 6.08
specificities and inhibitor tolerance despite their structural relationship.
Ó 2011 Elsevier Ltd. All rights reserved.
lM, respectively. These results suggest that NtMGAM and CtMGAM differ in their substrate
1. Introduction
In previous studies, the inhibition of MGAM and human pancre-
atic -amylase (HPA) activity showed a significant effect in control-
a
As the main dietary carbohydrate source for human consump-
tion, starch plays a very important role in human life. However,
excessive starch consumption or other carbohydrate-rich diets
can cause high glucose levels in blood and result in chronic dis-
eases like type 2 diabetes and obesity.1,2 In humans, four enzymes
are involved in the complete digestion of starch into glucose. Sali-
ling blood glucose levels making them prefect targets for the
treatment of type 2 diabetes and obesity.1,4,9 Acarbose, miglitol,
vogliboseand 1-deoxynojirimycin (DNJ) are commercially-available
glucosidase inhibitors with carbohydrate structures widely used in
the treatment of type 2 diabetes (Fig. 2). There are many acarvio-
sine-containing
a-amylase inhibitors that have been studied,
vary and pancreatic
a-amylases (EC 3.2.1.1) are endohydrolases
including acarbose, which display strong inhibitory potencies.10
Acarbose is a pseudo-tetrasaccharide composed of an acarviosine
that cleave the internal
a-(1?4) bonds of starch into shorter linear
and branched dextrin chains. The resultant mixture of dextrin is
then further hydrolyzed at the nonreducing ends into glucose by
two small-intestinal brush-border exohydrolases: maltase gluco-
amylase (MGAM; EC 3.2.1.20 and 3.2.1.3) and sucrose isomaltase
(SI; EC 3.2.148 and 3.2.10).3–6 MGAM is membrane-bound enzyme,
each containing two independent catalytic domains: an N-terminal
domain (NtMGAM) that is proximal to the membrane-bound end
and a C-terminal luminal domain (CtMGAM) (Fig. 1).7,8 These
two catalytic domains are 40% identical in amino acid sequence,
and all classified under glycosyl hydrolase family 31 (GH31).
group a-(1?4)-linked to a maltose (Fig. 2). Our group has isolated
several aminooligosaccharide natural metabolic products of Strepto-
myces coelicoflavus ZG065611 named as acarviostatins 1-03, 2-03,
3-03, and 4-03 (referred to as A1-03, A2-03, A3-03, A4-03), which
displayed much higher inhibitory activity against porcine
a-amy-
lase.12 They have a repeating pseudotrisaccharide unit that consists
of an acarviosine unit a-(1?4) linked to a glucose (Fig. 2). Especially,
A3-03 is the most potent inhibitor against PPA known to date.12
Moreover, they showed a significant effect in depressing blood
glucose levels in mammalian systems and could be developed to
possible therapeutic agents in the future..13
In this work, we first report the high quality and quantity clon-
ing, expression and purification of human CtMGAM as well as
⇑
Corresponding author. Tel.: +86 22 23508371.
E-mail address: baifang1122@nankai.edu.cn (F. Bai).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.09.012

L. Ren et al. / Carbohydrate Research 346 (2011) 2688–2692
2689
2. Results and discussion
MGAM is an important inhibition target in the treatment of type
2 diabetes and accounts for both glucoamylase activity and mal-
tase activity.2,7 Previous research has focused mainly on the N-ter-
minal domain of MGAM after the successful expression of human
NtMGAM in Drosophila cells.7 The mouse CtMGAM has recently
been cloned and expressed in Sf9 insect cells,1 CtMGAM was con-
sidered to have greater catalytic efficiency than NtMGAM and to be
responsible for the ‘glucoamylse’ activity of MGAM due to its high
affinity for larger oligoglucans.2 However, studies of CtMGAM, the
most important catalytic domain of MGAM are far behind NtM-
GAM and HPA. In this paper, we report the first expression of hu-
man CtMGAM in the Pichia pastoris expression system. This
allowed us to be the first to study human NtMGAM and CtMGAM
independently at the same time.
2.1. Expression and purification of NtMGAM and CtMGAM in
Pichia pastoris
The recombinant NtMGAM and CtMGAM proteins we
constructed cover amino acids from 87 to 954 and from 960 to
1853 of full length human MGAM. They were fused to an N-termi-
nal strep II tag and a C-terminal hexahistidine tag (Fig. 1). The
expected size of both proteins was around 110 KD. The recombi-
nant proteins were secreted to BMMY medium while being
induced by methanol. The concentration of methanol was up to
1%. The proteins were harvested after three days and the secreted
proteins were purified using Ni Sepharose resin from the BMMY
media followed by anion exchange chromatography and size-
exclusion column. Notable here was the high glycosylation degree
of recombinant proteins due to the expression system. Deglycosy-
lation was carried out by PNGase F using a size-exclusion column.
The purity of both CtMGAM and NtMGAM proteins was up to 99%
as analyzed by SDS–PAGE (Fig. 3). The total yield of pure recombi-
nant NtMGAM and CtAMGM proteins were about 2–3 mg/L.
Figure 1. The schematic structure of MGAM and the construction of recombinant
NtMGAM and CtMGAM protein inserted into pPIC9k expression vector. Features of
MGAM include the following: a transmembrane domain (TMD; ꢀ20 amino acids),
an O-glycosylated linker (O-link; ꢀ55 amino acids), and two homologous catalytic
domains (NtMGAM and CtMGAM; each ꢀ900 amino acids). Strep II and His6
indicate strep II tag and hexahistidine tag, respectively.
NtMGAM in the Pichia pastoris expression system for kinetic and
inhibition studies. Kinetic studies of DNJ, miglitol, voglibose, and
acarbose along with a series of acarviostatins against NtMGAM,
CtMGAM, and HPA were developed. The results provide us valuable
insight into the inhibitory mechanism of MGAM and HPA and will
benefit future anti-diabetic drug design.
OH
OH
OH
OH
OH
OH
NH
N
HO
HO
HO
HO
N
H
HO
HO
OH
OH
OH
Miglitol
DNJ
Voglibose
OH
OH
OH
OH
O
O
O
HO
HO
HO
HO
HO
HO
N
H
O
O
HO
HO
HO
Acarbose(A1-01)
OH
OH
OH
OH
OH
OH
O
O
O
O
O
HO
HO
HO
HO
HO
O
HO
O
O
n
N
H
O
HO
HO
HO
HO
HO
HO
HO
Acarviostatin 1-03(A1-03) :n=1
Acarviostatin 2-03(A2-03) :n=2
Acarviostatin 3-03(A3-03) :n=3
Acarviostatin 4-03(A4-03) :n=4
Figure 2. The structures of inhibitors mentioned in this paper: miglitol, voglibose, DNJ, acarbose, A1-03, A2-03, A3-03 and A4-03.

2690
L. Ren et al. / Carbohydrate Research 346 (2011) 2688–2692
GAM with Ki values of 1.14, 3.98, and 2.22
2).
lM, respectively (Table
In contrast to NtMGAM, all these
in this paper showed inhibitory activities against CtMGAM with Ki
values in range from 1.72 to 171.57 M (Table 2). The inhibitory
ability of acarbose, DNJ, and voglibose were at almost the same le-
vel, with Ki values of 1.72, 2.04, and 3.31 M, respectively. In addi-
a-glucosidase inhibitors used
l
l
tion, A1-03, A2-03, A3-03, A4-03 have shown high inhibitory
ability against CtMGAM with Ki values in the range of 6.02–
19.18 lM. However, miglitol was a very poor inhibitor against
CtMGAM. These inhibitors were all competitive inhibitors against
NtMGAM or CtMGAM.
The current antidiabetic compound, acarbose, was found to be a
micromolar inhibitor of the human NtMGAM (Ki = 51.88
contrast, acarbose is a 100-fold better inhibitor of human CtMGAM
(Ki = 1.72 M) and 1000-fold better for mouse CtMGAM-N2
(Ki = 0.009 M), CtMGAM-N20 (Ki = 0.028
M).1 This supports the
lM). In
l
l
l
notion that the tetrasaccharide analog occupies extended binding
subsites in the C-terminal catalytic subunits as compared to the
N-terminal domains.
Figure 3. The image of SDS–PAGE showed the molecule weight and the purity of
NtMGAN and CtMGAM proteins after S 200 column.
2.3.2. Inhibition against HPA
2.2. Catalytic studies of CtMGAM and NtMGAM
The catalytic and inhibitory mechanism of HPA, an endoglycosi-
dase, has been studied in a multifaceted way.14–19 In our previous
To compare the substrate specificities of NtMGAM and CtM-
GAM, we analyzed Km and Kcat values with maltose and soluble
starch. When maltose was used as the substrate, the Km values of
NtMGAM and CtMGAM were about 6.17 and 5.53 mM, respectively
(Table 1, and no obvious affinity difference was observed between
NtMGAM and CtMGAM. The Kcat of NtMGAM was 47.76 sꢁ1, which
is about two times that of CtMGAM (21.99 sꢁ1). These results
indicated that both catalytic domains of MGAM have maltose
digestion activities; however, NtMGAM was more potent in
degrading the shorter maltose substrate than CtMGAM.
work, acarviosine-containing
a-amylase inhibitors have shown
tremendous inhibitory effect on HPA. Compounds A2-03 and A3-
03 have displayed 170–180 times higher inhibitory activity than
acarbose.2 Their inhibition types were mixed. These results show
HPA has a very clear preference for inhibitors with longer sugar
chains than acarbose (AI-01).
In this research, DNJ and miglitol were found to be non-compet-
itive inhibitors against HPA, indicating the molecules might be too
small to have enough affinity to bind in the HPA active site alone.
As expected, their inhibitory ability against HPA was very low,
which was only 2% and 8% compared to acarbose. Additionally,
as we observed, voglibose was unable to inhibit HPA.
Because of the uncertainty of the soluble starch molecular
weight, we used the relation of its mass to its volume (percentage)
to represent the concentration of the substrate. The Km value of
CtMGAM was about twice that of NtMGAM when soluble starch
was used as substrate, and the Kcat was determined to be
We were interested in finding the best inhibitors against MGAM
and HPA in this research. In the inhibition analysis, aside from
acarbose, DNJ and miglitol were proven to have a broad spectrum
of inhibition effect on these glycosidases (NtMGAM, CtMGAM and
213.84 sꢁ1, thirty times higher than that of NtMGAM (6.82 sꢁ1
)
(Table 1). Evidently CtMGAM has starch digestion activity and
NtMGAM has little -glucosidase activity against complex starch
HPA), which catalyze the hydrolysis of a-(1?4)-glycosidic linkages
a
in starch digestion. No particular inhibitor was found to be a very
good inhibitor of all three targets. Among all inhibitors tested in
this study, the Ki of DNJ for NtMGAM and CtMGAM were small,
suggesting that DNJ had the strong ability to ‘brake’ the entire cat-
alytic ability of MGAM. In addition, A2-03 and A3-03 were not only
two of the strongest inhibitors against HPA, but also effective
inhibitors against CtMGAM, which will make them very valuable
in future drug design. In summary, to effectively block the pathway
of starch digestion, it is important to restrain both the beginning
and the end process of starch digestion to which the main targets
are referred to HPA and MGAM. According to our results, at least
molecules. These results suggest that NtMGAM and CtMGAM differ
in their substrate specificities and confirm the previous assump-
tion that CtMGAM might have glucoamylase activities.1,2
2.3. Inhibition analysis
2.3.1. Inhibition against NtMGAM and CtMGAM
From the inhibition studies of NtMGAN, acarbose displayed
poor inhibitory ability against NtMGAM with
51.88 M (Table 2). This result was consistent with a previous re-
sult, which showed that acarbose inhibited NtMGAM with a Ki of
62
M.7 Furthermore, A1-03, A2-03, A3-03, A4-03 failed to show
a Ki value of
l
two
a-glucosidase inhibitors were required to efficiently inhibit
l
the activities of MGAM and HPA. The combination of A2-03 (or
A3-03) and DNJ may potentially have stronger inhibitory effects
than a single inhibitor.
inhibitory activity against NtMGAM. However, we found DNJ, migl-
itol and voglibose had significant inhibitory activities against NtM-
Interestingly, out results reveal both NtMGAM and CtMGAM
have the ability of digesting soluble starch to produce glucose
and that CtMGAM has more significant efficiency than NtMGAM.
In comparison to acarbose, A2-03 and A3-03 display much higher
inhibitory activities against HPA, lower inhibitory activity against
CtMGAM, and failed to inhibit NtMGAM. The selectivity of the
two human MGAM catalytic domains toward different inhibitors
was also reflected in the report as mouse CtMGAM-N2 and N20
were more sensitive to the longer inhibitors than human ntM-
GAM.1 We propose that the explanation of these phenomena
Table 1
Substrate kinetics of NtMGAM and CtMGAM
Enzyme
Maltose
Kcat (Sꢁ1
Soluble starch
Kcat (Sꢁ1
)
Km (mM)
)
Km (mass/
volume)
NtMGAM 6.17 0.66 47.76 10.34 1.18% 0.42%
CtMGAM 5.53 0.32 21.99 1.25 2.72% 1.45%
6.82 1.35
213.84 43.42

L. Ren et al. / Carbohydrate Research 346 (2011) 2688–2692
2691
Table 2
Inhibition of acarbose, A1-03, A2-03, A3-03, A4-03, DNJ, miglitol, voglibose against HPA, NtMGAM, and CtMGAM
Inhibitor
HPA
NtMGAM
Inhibition type
CtMGAM
Inhibition type
Ki (
lM)
Ki (
lM)
Inhibition type
Ki (lM)
Acarbose
A1-03
A2-03
A3-03
A4-03
2.59 0.010a
1.25 0.030a
0.015 0.0012a
0.014 0.00020a
0.47 0.0096a
Competitive
51.88 3.06
Competitive
Competitive
Competitive
Competitive
Competitive
1.72 0.05
12.20 2.05
6.02 0.68
6.08 0.18
19.18 2.43
Mixeda
Mixeda
Mixeda
Mixeda
—
—
—
—
—
—
—
—
Mixeda
DNJ
Miglitol
Voglibose
Non-competitive
Non-competitive
—
122.36 10.18
31.50 6.50
—
Competitive
Competitive
Competitive
1.41 0.16
3.98 0.04
2.22 0.26
Competitive
Competitive
Competitive
2.04 0.19
171.57 13.39
3.31 0.21
Ki is the inhibition constant, defined as [E][I]/[EI].
—: no significant inhibition was observed.
a
Data from Qin and Ren.3
may lie in the structure of the active cleft of these glucosidases.
Relatively small inhibitors such as DNJ, miglitol, voglibose dis-
played tremendous inhibitory effect on NtMGAM suggesting a rel-
atively small catalytic cleft, which is consistent with the
observation of the active cleft in the crystal structure of NtMGAM.4
vectors were electroporated into Pichia pastoris (GS115). The positive
strains were obtained though G418 screening in 96-well plates and
confirmed by dot-blotting using the antibody of his6. Recombinant
NtMGAM and CtMGAM proteins were secreted into BMMY medium
while being induced with 1% methanol. The proteins were harvested
between 72 and 96 h.
On the other hand, acarviosine-containing
a-amylase inhibitors
including acarbose have shown strong inhibitory ability against
CtMGAM and HPA indicating that they might have a larger cata-
lytic cleft. This was confirmed by our previous studies on the crys-
tal structure of HPA in complex with acarviostatins.3 Additionally,
all the inhibitors used in this paper had the inhibitory ability
against CtMGAM, further indicating that it may have a flexible
and adaptable active cleft.
Ni Sepharose™ 6 Fast Flow resin (GE healthcare) was used to
purify the secreted NtMGAM and CtMGAM proteins from BMMY
medium (10–15 ml resin/L media). Resin was pre-equilibrated by
20 mM Tris–HCl, pH 7.0 containing 200 mM NaCl. The recombinant
proteins bound to the resin overnight at 4 °C. After being washed
by pre-equilibration buffer, the proteins were eluted by 20 mM
Tris–HCl pH 7.0, contains 200 mM NaCl and 300 mM imidazole.
The eluted fractions were concentrated and dialyzed against
20 mM Tris–HCl, pH 7.0, which is also the start and equilibration
buffer of HiTrap Q HP anion exchange column (GE Healthcare).
Then, the recombinant proteins were eluted with a linear gradient
of 0–500 mM NaCl. The fractions were concentrated to 2 mL and
deglycosylated by PNGase F overnight at room temperature. Final-
ly, a Superdex S 200 size-exclusion column (GE Healthcare) was
used to further purify NtMGAM and CtMGAM. The S 200 column
was pre-equilibrated with the running buffer 20 mM Tris–HCl,
pH 7.0, containing 200 mM NaCl. The proteins obtained were up
to 99% pure as analyzed by SDS–PAGE.
3. Experimental
3.1. General methods
All chemicals and buffers salts used were from Sigma unless
otherwise indicated. The expression and purification of recombi-
nant HPA was performed as described previously.2 HPA was dia-
lyzed against 20 mM potassium phosphate buffer, with pH 6.9
and the protein concentration was 2 mg/mL. In the enzyme kinetic
studies, reactions were performed in 96-well plates in triplicate.
Due to the different optimal buffers for each enzyme, substrates
and inhibitor were diluted in the buffer 100 mM sodium acetate
trihydrate, pH 4.8 for NtMGAM or 20 mM Tris–HCl, pH 7.0 for
CtMGAM.
3.3. Kinetic assays of CtMGAM, NtMGAM and HPA
Km values of NtMGAM and CtMGAM were determined by mea-
suring the production of glucose (Glu Kit, Biosino Bio-technology
and Science Inc.) using maltose and soluble starch as substrates.
The catalytic rate constant (Kcat) values of NtMGAM and CtMGAM
were determined by the number of glucose that was converted
from each NtMGAM or CtMGAM substrate per unit time, and in
which the enzyme was at its maximum efficiency. The reaction
3.2. Cloning, expression and purification of NtMGAM and
CtMGAM
Recombinant NtMGAM and CtMGAM proteins were expressed in
the Pichia pastoris protein expression system. The NtMGAM and
CtMGAM cDNA were cloned from pReceiver-Y01 vector contain
the cDNA of full length human MGAM (Genbank Accession
NM_004668.1 from GeneCopoeia). NtMGAM and CtMGAM contain
residues from 87 to 954 and from 960 to 1853 of full length human
MGAM. NtMGAM was amplified by the upstream primer 50-AAG-
GAAAAAAcctaggTGGAGCCACCCGCAGTTCGAAAAG tctgctgaatgtccag
tgg-30 and downstream primer 50-AAGGAAAAAA cctaggATGATGG
TGATGGTGATGccttatctttatgctccattcc-30. CtMGAM were amplified
by the upstream primer 50-AAGGAAAAAAcctaggTGGAGCCACC
mixture contained 10
and 10 L maltose (1–10 mM). The reactions were carried out at
37 °C for 30 min. 20 L 2 M Tris–HCl, pH 7.0 was used to stop
the reaction, then the reactions were developed with 150 L Glu
lL NtMGAM (0.2 lM) or CtMGAM (0.2 lM)
l
l
l
kit reagent at 37 °C for 15 min to determinate the amount of glu-
cose produced. Absorbance was measured at 490 nm in a bio-rad
microplate spectrophotometer. The volume of reaction mixture
was 200
mixture contains 150
GAM (1 M) or 50
37 °C for 30 min, the reactions were stopped by 200
HCl pH, 7.0. 40 L samples were taken to determine the glucose le-
l
L when the substrate was soluble starch. The reaction
L soluble starch (0.1–1%) and 50 L NtM-
L CtMGAM (0.1 M). After incubation at
L 2 M Tris–
l
l
l
CGCAGTTCGAAAAG gatgaagaaaaaatagactgttac-30
and
down-
l
l
stream primer 50-AAGGAAAAAAcctaggATG ATGGTGATGGTGATGccac
gtcaatgaagtaaaattatg-30. The PCR products were cloned into pPIC9k
expression vector by Avr2 site. After being linearized by SalI and SacI,
respectively, recombinant pPic9k-NtMGAM and pPic9k-CtMGAM
l
l
vel. The following procedures were the same as we mentioned
above.

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L. Ren et al. / Carbohydrate Research 346 (2011) 2688–2692
The inhibitory constants (Ki) values of miglitol, voglibose, DNJ,
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GAM (0.2 M) were added to 10 L mixture of maltose (1–10 mM)
with inhibitors of different concentrations (0.5–40 M). The reac-
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l
l
l
HCl, pH 7.0 was used to stop the reaction. The glucose detection
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This work was supported by the National Natural Science Foun-
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Development Program of China (973 Program) (2007CB914803),
the Tianjin Municipal Science and Technology Commission of Chi-
na (10JCYBJC14300) and the Fundamental Research Funds for the
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Supplementary data associated with this article can be found, in
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