Maltase Decorated by Chiral Carbon Dots with Inhibited Enzyme Activity for Glucose Level Control

Article abstract of DOI:10.1002/smll.201901512

Carbon dots (CDs) have attracted increasing attention in disease therapy owing to their low toxicity and good biocompatibility. Their therapeutic effect strongly depends on the CDs structure (e.g., size or functional groups). However, the impact of CDs chirality on maltase and blood glucose level has not yet been fully emphasized and studied. Moreover, in previous reports, chiral CDs with targeted optical activity have to be synthesized from precursors of corresponding optical rotation, severely limiting chiral CDs design. Here, chiral CDs with optical rotation opposite to that of the precursor are facilely prepared through electrochemical polymerization. Interestingly, their chirality can be regulated by simply adjusting reaction time. At last, the resultant (+)-DCDs (700 μg mL^?1) are employed to modify maltase in an effort to regulate the hydrolytic rate of maltose, showing an excellent inhibition ratio to maltase of 54.7%, significantly higher than that of (?)-LCDs (15.5%) in the same reaction conditions. The superior performance may be attributed to the preferable combination of DCDs with maltase. This study provides an electrochemical method to facilely regulate CDs chirality, and explore new applications of chiral CDs as antihyperglycemic therapy for controlling blood glucose levels.

Full text of DOI:10.1002/smll.201901512

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COMMUNICATION
Chiral Carbon Dots
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Maltase Decorated by Chiral Carbon Dots with Inhibited
Enzyme Activity for Glucose Level Control
Mengling Zhang, Huibo Wang, Bo Wang, Yurong Ma, Hui Huang, Yang Liu,*
Mingwang Shao,* Bowen Yao,* and Zhenhui Kang*
but with a lower efficacy than biguanides
and sulfonylureas.[3,5,7] Therefore, it
Carbon dots (CDs) have attracted increasing attention in disease therapy
owing to their low toxicity and good biocompatibility. Their therapeutic effect
strongly depends on the CDs structure (e.g., size or functional groups).
However, the impact of CDs chirality on maltase and blood glucose level
has not yet been fully emphasized and studied. Moreover, in previous
reports, chiral CDs with targeted optical activity have to be synthesized
from precursors of corresponding optical rotation, severely limiting chiral
CDs design. Here, chiral CDs with optical rotation opposite to that of the
precursor are facilely prepared through electrochemical polymerization.
Interestingly, their chirality can be regulated by simply adjusting reaction
time. At last, the resultant (+)-DCDs (700 µg mL−1) are employed to modify
maltase in an effort to regulate the hydrolytic rate of maltose, showing an
excellent inhibition ratio to maltase of 54.7%, significantly higher than that of
(−)-LCDs (15.5%) in the same reaction conditions. The superior performance
may be attributed to the preferable combination of DCDs with maltase. This
study provides an electrochemical method to facilely regulate CDs chirality,
and explore new applications of chiral CDs as antihyperglycemic therapy for
controlling blood glucose levels.
is necessary to develop
a new inhi-
bitor with a high inhibitory efficacy for
antihyperglycemic.
Carbon dots (CDs), as an emerging
subclass of carbon materials, having been
applied in various biological applica-
tions, including bioimaging, drug/gene
delivery, antimicrobial and plant biology,
owing to their low toxicity, well biocom-
patibility, and biodegradable property.[8–10]
For different purpose, CDs have been
reported to combine with various drug
or biomacromolecules, such as (1) CDs/
protoporphyrin IX composite for nucle-
olus imaging and nucleus-targeted drug
delivery owing to their selectively targeting
nucleolus ability[11]; (2) CDs/ssDNA com-
posite as ideal fluorescent sensing plat-
form for nucleic acid detection[12]; (3) In
addition, CDs can be biodegraded to plant
hormone analogues and CO2 through the
horseradish peroxidase of plants, demon-
Nowadays, diseases arisen from high blood glucose level have
grown explosively around the world, including type 2 diabetes,
hyperglycemia, and some cardiovascular diseases.[1,2] Besides, a
high blood glucose level also severely endangers those people
with impaired glucose tolerance[3,4] Therefore, controlling
blood glucose level is critical in the process of therapy. There
are several types of oral antihyperglycemic drug for decreasing
blood glucose level, such as biguanides, sulfonylureas, and
α-glucosidase inhibitor. Among these medicines, biguanides
and sulfonylureas can efficiently decrease high blood glucose
but usually accompanied by serious side effects,[5,6] whereas
α-glucosidase inhibitors (e.g., acarbose and miglitol) are safer
strating its excellent biocompatibility.[13]
Chirality is a common phenomenon, and takes critical role
in physiological activities.[14,15] Therefore, the CDs chirality are
also expected to strongly affect their biological properties. For
instance, l-CDs prepared from l-cysteine showed upregulated
glycolysis in cellular energy metabolism of human bladder
cancer T24 cells, but d-CDs showed no effect[16]; l-CDs synthe-
sized from l-lysine dramatically remodeled Aβ42 structure and
inhibited Aβ42 cytotoxicity, compared with d-CDs[17]
; d-CDs
obtained from d-glutamic acid have more efficient antimicro-
bial activity towards both Gram-negative and Gram-positive
bacteria than l-CDs[18]; Besides, the different chirality of CDs
can even showed opposite effects. For example, the l-CDs syn-
thesized from l-cysteine can improve laccase activity, whereas
its counterpart d-CDs impair the activity.[19] However, the effect
of chiral CD on maltase and blood glucose level was rarely
reported. Besides, according to the previous reports, chiral CDs
of targeted optical activity have to be synthesized from pre-
cursor of corresponding optical rotation (e.g., l-CDs must be
prepared from sinistral precursor), severely limiting the strate-
gies to synthesize chiral CDs. Chiral CDs with optical rotation
opposite to that of precursor have barely been reported.
M. Zhang, H. Wang, B. Wang, Y. Ma, Prof. H. Huang, Prof. Y. Liu,
Prof. M. Shao, Dr. B. Yao, Prof. Z. Kang
Institute of Functional Nano & Soft Materials (FUNSOM)
Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices
Soochow University
199 Ren’ai Road, Jiangsu, Suzhou 215123, P. R. China
E-mail: yangl@suda.edu.cn; mwshao@suda.edu.cn; bwyao@suda.edu.cn;
zhkang@suda.edu.cn
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201901512.
Herein, chiral CDs were prepared by electrochemical poly-
merization from l- or d-glutamic acid (Glu) in aqueous alkali, and
DOI: 10.1002/smll.201901512
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their optical activity was tuned facilely by controlling the oper-
ating time of preparation. Interestingly, the circular dichroism
(CD) optical orientation could even transform (e.g., from sinis-
tral to dextral) after simply prolonging the reaction times to
6 d. Finally, their inhibitory efficiencies on maltase activity were
studied in terms of inhibition ratio and Michaelis constant
(Km and Vmax) by using maltase and maltase/CDs hybrids as
enzyme and maltose as substrate. The superior performance
may be attributed to the preferable combination of DCDs with
maltase. To sum up, this study provides a new method to regu-
late CDs chirality, and develop chiral CDs as antihyperglycemic
therapy for controlling blood glucose level.
The chiral CDs were synthesized by electrochemical poly-
merization from l- or d-Glu in aqueous alkali (Figure 1). Consi-
dering the low solubility of l- or d-Glu in water solution, the pH
of the solution was increased by adding NaOH to enhance their
solubility and increase the electrical conductivity of electrolyte
during the electrochemical synthesis.[19] The chiral CDs ((+)-
LCDs or (−)-DCDs) could be obtained after 3 d and their optical
activity were inherited from l-(+) or d-(−)-Glu. Interestingly, the
optical activity of CDs was reversed after electropolymerization
for 6 d. When l-(+)-Glu or d-(−)-Glu were used as precursor, (−)-
LCDs or (+)-DCDs (hereinafter called as LCDs and DCDs) were
obtained. The text mainly discussed DCDs and the characteri-
zation of LCDs are provided in the Supporting Information.
The morphologies of chiral CDs were obtained by transmis-
sion electron microscopy (TEM) and atomic force microscopy
(AFM). As shown in Figure 2a, DCDs were well dispersed,
and their diameter ranged from 2 to 6 nm. The high-resolu-
tion transmission electron microscopy (HRTEM, the inset
of Figure 2a) revealed the lattice spacing of 0.21 nm, corre-
sponding to the (100) crystal facet of graphite.[20] As shown in
AFM images, DCDs had a circle shape with the topographic
height of 3–7 nm (Figure S1, Supporting Information),
agreeing well with the results of TEM characterization. The
AFM and TEM images of LCDs were shown in Figures S1 and
S2 in the Supporting Information.
Figure 1. Schematic for the synthesis of chiral CDs.
UV–vis absorption spectrum, photoluminescence (PL) spec-
trum, and CD spectrum were measured to study the optical
properties of chiral CDs. For the UV–vis absorption of l-Glu
(Figure S5, black line, Supporting Information), only one
absorption peak located at 208 nm was observed in the absorp-
tion curve, which corresponded to π–π* transitions.[26] After
CDs were formed, the π–π* transitions showed a red shift from
208 to 218 nm as the result from the expanded conjugated
domains,[27] and a new n–π* transitions (the peak located at
283 nm) appeared (Figure 2d, black line; and Figure S5, blue
line, Supporting Information).[26,28] PL spectrum of chiral CDs
was also detected (Figure 2d; Figure S6, Supporting Informa-
tion). DCDs solution revealed an optimal emission at 405 nm
(Figure 2d, red line) with 325 nm excitation (Figure 2d, blue
line). The CD spectrums of chiral CDs were also measured
for monitoring the formation of CD chirality. As illustrated in
Figure 2e, the chiral signals of CDs prepared at different elec-
trolysis time were measured. When the solution was electro-
lyzed for 1 d, the CD signal located at 205 nm. With the electrol-
ysis time increased to 2 d, the intensity of CD signal at 205 nm
significantly increased. The CD signal of precursors (Figure S7,
Supporting Information) was at 205 nm as well. Hence, the CD
signal located at 205 nm from chiral CDs should be inherited
from l- or d-Glu. However, when the electrolysis time extended
to 3 d, the intensity of CD signal decreased. Then, since the
electrolysis time was prolonged to 4 d, the CD signal of solu-
tion disappeared. Interestingly, the signal does not vanish with
the increase of electrolysis time to 5 and 6 d, but a reverse CD
signal of the solution appeared at the same position and the
intensity of this signal gradually increased. A pair of chiral CDs
with opposite optical rotation to the raw materials was obtained
The elementary composition and chemical structures of CDs
were analyzed by X-ray photoelectron spectroscopy (XPS) and
Fourier transform infrared (FTIR) spectrum. The full scan XPS
spectrum of DCDs and LCDs were shown in Figures S3a and S4a
(Supporting Information), which showed the CDs were mainly
composed by C, N, and O elements. For DCDs, C, N, and O
accounted for 58.40, 7.49, and 34.11 at%. The C 1s XPS spectrum
of DCDs (Figure 2b) could be deconvoluted into three peaks at


284.60, 286.00, and 287.80 eV, corresponding with C C, C O/
C N, and C O.[21,22] One surface components of DCDs could be
found in the N1s deconvoluted spectra and the peak at 399.50 eV
was belonged to the pyridinic N (Figure S3b, Supporting Informa-
tion).[21,23] The analysis results of XPS for LCDs were displayed
in Figure S4 in the Supporting Information. The FTIR spectrum
was employed for analyzing the surface functional groups of the
obtained chiral CDs (Figure 2c). The vibrational absorption signals


were less than raw materials (black line). The peak at 3446 cm−1
[24]


was corresponded to the stretching vibration of O H or N H.
The vibrational absorption signals at 1578 and 1390 cm−1 could be
[21,25]


attributed to the vibration of C C and C O.
Furthermore,
FTIR spectrum of LCDs (blue line) and DCDs (red line) showed
similar functional groups, such as −NH and −OH.
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Figure 2. Characterization of CDs. a) TEM image and b) XPS spectrum of DCDs; c) The FTIR spectrum of LCDs (blue line), DCDs (red line), and l-Glu
(black line); d) UV–vis spectrum (black line), PL excitation (blue line), and emission spectrum (red line) of DCDs; e) The CD spectrum of LCDs (blue
line) and DCDs (red line) prepared at 1–6 d, respectively.
through a facile alkaline-assisted electrochemical polymeriza-
tion from l- and d-Glu.
The concentration of maltase and LCDs or DCDs were
3.6 mg mL−1 and 700 µg mL−1. Throughout the first 30 min
after the reaction started, the glucose generation rate was pro-
portional to the reaction time. When the reaction time reached
to 90 min, the enzymatic reaction reached equilibrium. During
3 h of the reaction, it was obvious that both the LCDs and
DCDs inhibited the activity of maltase and the DCDs had better
inhibitory effect than the LCDs. For better understanding the
influence of different concentration of chiral CDs in the cata-
lytic activity of maltase/LCDs or maltase/DCDs, the inhibi-
tion ratio of chiral CDs with different concentration (200, 400,
700, 1000, 1500 µg mL−1) were detected (Figure 3b) when the
reaction reached equilibrium (90 min). With the increasing
concentration, the inhibition ratio of maltase/LCDs (blue trace)
and maltase/DCDs (red trace) gradually enhanced. Remarkably,
in the concentration of CDs ranging from 200–1500 µg mL−1
in maltase/LCDs or maltase/DCDs hybrids, DCDs had more
effective in inhibiting the activity of maltase. That is to say, the
chirality of the CDs had an impact on the activity of maltase.
Maltase is a key enzymes involved in carbohydrates diges-
tion and it can catalyzes the hydrolysis of maltose to the blood
glucose in human body.[7,29] Controlling blood sugar level has
attract the attention of researchers with the improving of living
standard.[1] Before biological application of the chiral CDs, the
cytotoxicity and biocompatibility of chiral CDs was evaluated
via 293T cells at first, which was incubated with CDs with an
increasing concentration. As shown in Figure S8 (Supporting
Information), the chiral CDs did not affect the growth and pro-
liferation of the 293T cells even at an ultrahigh concentration
(2.4 mg mL−1). This data demonstrated the chiral CDs were low
toxicity and benign biocompatibility.
In the following experiment, the chiral CDs with oppo-
site optical rotation to the raw materials were combined with
maltase for tuning activity. Figure 3a showed the glucose con-
centration versus different reaction times for maltase (black
line), maltase/LCDs (blue line), and maltase/DCDs (red line).
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Figure 3. Maltase inhibitory activities of chiral CDs. a) The glucose concentration of the maltose degradation for maltase (black line), maltase/LCDs
(blue line), and maltase/DCDs (red line) at different hydrolysis times (0–180 min, the concentration of maltase and LCDs or DCDs is 3.6 mg mL−1 and
700 µg mL−1, respectively); b) The inhibition ratio of LCDs (blue trace) and DCDs (red trace) with different concentration (200–1500 µg mL−1) after
90 min reaction (maltase: 3.6 mg mL−1); c) Lineweaver–Burk plots of maltase (black line), maltase/LCDs (blue line), and maltase/DCDs (red line);
(m: mol L−1, m m: mmol L−1, Mean SD, ***P < 0.001).
To investigate how the chiral CDs inhibited the activity of
maltase, Michaelis–Menten kinetics analysis was employed and
the Michaelis constants (Km and Vmax) for maltase, maltase/
LCDs, and maltase/DCDs were obtained by Lineweaver–Burk
plot. Km and Vmax could be calculated from the ordinate and
abscissa intercept which represent the value of 1/Vmax and
−1/Km.[30,31] The Lineweaver–Burk plot of maltase (black line),
maltase/LCDs (blue line), and maltase/DCDs (red line) were
shown in Figure 3c and the value of Michaelis constants were
shown in Table S1 in the Supporting Information. The concen-
tration of maltase and LCDs or DCDs were 3.6 mg mL−1 and
700 µg mL−1. From the Lineweaver–Burk plot, the value of the
Vmax of maltase, maltase/LCDs, and maltase/DCDs were 1.16,
0.80, and 0.59 mmol L−1 min−1 and the Km of which were 0.25,
0.13, and 0.17 mol L−1, respectively. In the same reaction con-
ditions, both the Vmax and Km of maltase/LCDs or maltase/
DCDs decreased, which demonstrated the LCDs or DCDs was
a kind of mixed inhibitor for maltase.[31,32] Besides, comparing
maltase/LCDs with maltase/DCDs, the Vmax of maltase/LCDs
was greater than maltase/DCDs in the maltase concentration of
3.6 mg mL−1, indicating the maltase/LCDs had higher catalytic
efficiency than maltase/DCDs. For Km, which value is equal
was used for testing the hydrodynamic diameter of maltase
(black line) and maltase/CDs hybrids (maltase/LCDs: blue
line; maltase/DCDs: red line). As shown in Figure 4b, the size
of maltase/CDs hybrids were larger than free maltase. In the
hydrodynamic diameter of maltase/LCDs and maltase/DCDs, it
was more change in the size of the maltase/DCDs than which of
the maltase/LCDs. From the discussion above, it could be con-
firmed that the chiral CDs could be combined with maltase. For
investigating the interaction between maltase and chiral CDs
in maltase/CDs hybrids, PL spectrums of maltase (black line),
maltase/LCDs (blue line) and maltase/DCDs (red line) were
obtained (Figure 4c). The fluorescence intensity of maltase/CDs
is higher than free maltase and the emission peak is almost
unchanged (about 7 nm), illustrating the chiral CDs may be
combined with maltase by noncovalent bonds.[30] Then, the
secondary structure of maltase and maltase/CDs hybrids were
analyzed by CD spectrum (Figure 4d), compared with the CD
signals of free maltase (black line), the intensity of CD signals
of the maltase/LCDs (blue line) and maltase/DCDs (red line)
decreased at the negative peaks of 208, 222 nm (α-helix), and
218 nm (β-helices).[33,34] However, the intensity of CD signals for
maltase/DCDs was lower than that of the maltase/LCDs hybrid,
suggesting that the chiral CDs had changed the structure of
maltase and the structure of maltase treated with DCDs changed
more. Based on the above discussion, the possible inhibition
mechanism of chiral CDs for Maltose catalyzed reaction might
be as follows: the chiral CDs could be combined with maltase by
noncovalent bonds and then changed the structure of maltase.
They may occupy the active sites or other sites of maltase. And
then, the maltase/CDs hybrids likely hindered the combina-
tion of enzyme with substrates for reducing enzymatic activity.
Another possibility is that the maltase/CDs hybrids could com-
bine with substrates to form a maltase/CDs/maltose compound,
which could not release the products resulted in the low glucose
content. Besides, the DCDs could be preferably combined with
maltase and affected its activity compared with the LCDs.
to the concentration of substrate when the reaction rate reach
[31]
to the half of Vmax
.
In spite of the smaller Vmax of maltase/
DCDs compared with maltase/LCDs, the Km of maltase/DCDs
were larger than that of maltase/LCDs. It demonstrated the
weaker affinity of maltase/DCDs towards the maltose. Taking
the above discussions into consideration, the maltase/DCDs
had the higher inhibitory effect than maltase/LCDs hybrids.
To better confirm the combination of LCDs or DCDs with
maltase. Sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE) test, dynamic light scattering (DLS) detec-
tion, CDspectrum, andPLspectrumformaltaseandmaltase/CDs
hybrids were analyzed. The result of SDS-PAGE test was shown
in Figure 4a. The displacement distance of the maltase/LCDs
is shorter than free maltase, and the maltase/DCDs was slower
than maltase/LCDs and free maltase, which demonstrated that
the LCDs or DCDs successfully combined with maltase and the
DCDs combined more effectively with maltase than the LCDs.
For further demonstrating the successful combination, DLS
In summary, we have prepared chiral CDs by electrochemical
polymerization from l-(+)- and d-(−)-Glu in aqueous alkali. The
chirality of CDs could be regulated and even reversed by con-
trolling reaction time. With the increase of electrolysis time, the
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