A glucose-activatable trimodal glucometer self-assembled from glucose oxidase and MnO2 nanosheets for diabetes monitoring

Article abstract of DOI:10.1039/c7tb00864c

Daily monitoring of blood glucose is of great importance for the treatment of diabetes mellitus. Herein, we present an ensemble glucometer with a sandwich structure formed by the spontaneous entrapment of glucose oxidase (GOD) onto manganese dioxide nanosheets (MnO₂ NSs) via the hydrophobic effect and hydrogen bond interaction. Within the hybrid glucometer, the ultrathin MnO₂ NSs act as an enzyme nanosupport and target-activated signal transducer. Trimodal self-indication by fluorescence (FL) and UV-absorbance (UV) and magnetic resonance signal (MRS) activation with glucose-specificity provides multiple response signals to glucose. Taking account of its operational simplicity and convenience, even being observable by the naked eye, a detection limit as low as 0.1 ;M was obtained by using the ensemble glucometer in a colorimetric assay, whilst the precision for 11 replicated detections of 10 M glucose was 3.5% (relative standard deviation, RSD). Notably, the value of the Michaelis-Menton constant of GOD involved the presented glucometer is estimated to be 0.051 mM, showing an exceptional enhanced enzymatic activity of free GOD measured by far. The designed glucometer, with its high sensitivity and simplicity highlighted, was capable of routine blood glucose monitoring for type-I diabetes mellitus in rats. Furthermore, the fully integrated platform can be readily generalized in principle for a number of biomarkers for point of care diagnostics in the future.

Full text of DOI:10.1039/c7tb00864c

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Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
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A glucose-activatable trimodal glucometer self-assembled from
glucose oxidase and MnO nanosheets for diabetes monitoring
2
Received 00th January 20xx,
Accepted 00th January 20xx
abc†
ac†
ac
d
ac
ac
ac
Jin-Long Chen,*
Li Li, Shuo Wang, Xiao-Yan Sun, Lu Xiao, Jia-Shu Ren, Bin Di, and Ning
b
Gu*
DOI: 10.1039/x0xx00000x
Daily monitoring of blood glucose is of great importance for the treatment of diabetes mellitus. Herein, we presented an
ensemble glucometer with a sandwiched structure formed by the spontaneouse entrapment of glucose oxidase (GOD)
www.rsc.org/
onto manganese dioxide nanosheets (MnO
2
NSs) via hydrophobic effect andhydrogen bond interaction. Among of hybrid
glucometer, the ultrathin MnO NSs acts as an enzyme nanosupport and target-activated signal transduction. A trimodal
2
self-indicating by fluorescence (FL) and UV-absorbance (UV) and magnetic resonance signal (MRS) activation with glucose-
specificy provides a multiple signals of response to glucose. Taking account of operational simplicity and convenience even
observed by naked-eye, a detection limit low as 0.1 ꢀM was obtained by using of the ensemble glucometer in colorimetric
assay, whilst the precision for 11 replicate detection 10 ꢀM glucose was 3.5% (relative standard deviation, RSD). Notably,
the value of the Michaelis–Menton constant of GOD of the presented glucometer is estimated to be 0.051 mM, showing
an exceptional enhanced enzymatic activity measured by far. The designed glucometer highlighted with high sensitivity
and simplicity was capable of routine blood glucose monitoring for type I diabetes mellitus of rats. Furthermore, the fully
integrated platform is readily generalized in principle for a number of biomarkers for point of care diagnostic in the future.
variety of promising applications for stimuliꢀresponse drug
delivery and especially for targetꢀactivatable biosensor
Introduction
7
ꢀ9
4, 10ꢀ15
The continuous point of care testing of biomarkers gives unique
solutions for the treatment of chronic, homeostasisꢀrelated
in recent years. However, a powerful platform for biosensing
by integrating activatable mediator agents with susceptible
enzymes is still challenging free of requirements for either
sophisticated instrument and operation or much specific
knowledge. Notably, glucoseꢀGOD catalysis cascade reaction
as a proof of concept has extensively used in design of
1
,2
diseases such as diabetes mellitus. One strategy is the use of
multimodal probes response to specific biological species
which would help to maximize the signal from the target and
minimize background signal, improving sensitivity and
3
,4
specificity. Prominent among which is generally developed
biosensors for detection of glucose and many nonꢀglucose
5
,6
by integration of signals transduction with active enzymes.
16ꢀ21
targets by far.
Furthermore, the detection of blood glucose
However, one of the major obstacles for enzymeꢀinvolved
probes remains the lack of compatible approach for ensemble
susceptible native enzymes with signal transduction agents.
Recently, twoꢀdimensional nanomaterials have attracted high
attention owing to their ultimate structure and fascinating
has attracted continuous attention because diabetes mellitus is
one of the highest worldwide public health threats. One of the
major challenges in the management of these diabetic patients
is the frequent monitoring of blood glucose concentrations
because good diabetic control is very difficult to achieve.
Despite years of research, a variety of methods and techniques
22, 23
2
properties. Especially ultrathin MnO NSs with high specific
surface area appeared to act as benign cargoes to trap and
support drugs and biomolecules which have employed a wide
have been developed to detect the glucose concentrations,
24, 25
including nearꢀinfrared spectroscopy
, electrochemical
2
6
27, 28
biosensors and fluorescence assays
. However, most of
current methods still require expensive instrumentations and
tedious procedures as well as poor selectivity that are not
widely available to the general public for quantitative analysis.
In contrast, practical glucose monitoring should be accurate,
simple, and low cost as far as possible.
We herein presented a glucoseꢀspecific response glucometer
characterized with readout of multiple signals and high
enzymatic activity which was facile obtained just mixing of
GOD and MnO
2
NSs in aqueous solution at ambient
environment. Significantly different from previous methods and
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2
MnO NSs in various concentrations of glucose were acquired
from a 0.5 T MR scanner (MesoMR60, Shanghai Niumag
Corporation, China). The test parameters were as follow: multi
spinꢀecho, TR/TE =2000/60 ms, slices=1, matrix of 192×256
and FOV of 100×100 mm.
The morphology and microstructure of the MnO NSs were
2
characterized by transmission electron microscope (TEM) on a
Tecnai G2 F20 (Philips, Holland) operating at 200 kV
2
accelerating voltage. The height profile of MnO NSs was
studied by atomic force microscopy (AFM) (Multimode
Nanoscope IIIa controller) under tapping mode on a mica
substrate. Fourier transform infrared (FTꢀIR) spectra were
measured from a KBr window on a FT/IRꢀ4100 JASCD
spectrophotometer. Circular dichroism (CD) spectra were
recorded with a Jꢀ810 (JASCD). Inductively coupled plasma
Fig.1 Schematic illumination for an ensemble glucometer with sandwiched structure
self-assembled spontaneously from GOD and MnO
2
NSs characterized with an
exceptional enhanced enzymatic activity as well as a self-indicating of glucose with
readout of three channel signals of UV/MR/FL.
technologies for glucose detection using of GOD and MnO
(
2
Fig.1), 10, 13, 15a selfꢀindicating approach via direct triple optical emission spectrometry (ICPꢀOES) Optima 5300DV was
4,
signal readout was firstly developed superior to those glucose used to analysis concentration of MnO NSs as manganese ion.
2
biosensors with only readout of either single channel or doubleꢀ Commercially available blood glucose meter (GAꢀ3, SANNUO)
channel signals in complex matrixes. Notably, an unusual was applied to detect of blood glucose collected from SD rats.
enhancement of the enzymatic activity of GOD was observed
Preparation of MnO NSs with the assistant of SDS
2
which endowed the ensemble glucometer with high sensitivity
favorable for detection of blood glucose especially for Colloidal MnO
colorimetric assay. Furthermore, a diversity of hybrid platforms the assistant of SDS in a aqueous acidic solution according to a
nanosheets were synthesized by oneꢀstep with
2
2
9
is readily generalized in principle by integrated MnO
2
with modified method. Briefly, a diluted H
2
SO
4
solution (1.8 M,45
numerous biomolecules for the diagnostics and treatments of
diseases in the future.
ꢀ
L) and SDS (461.4 mg) were sequentially added into 160 mL
distilled water and heated at 95
℃ for 15 min by first, as
followings, the temperature of the mixture solution was reduced
to 85 . KMnO solution (0.05 M,1.6 mL) was rapidly add to
the above SDS solution and started to reaction under stirring at
85 for about 35 min to obtain a dark brown homogeneous
℃
Experimental section
4
Materials and Chemicals
℃
solution (Fig. S1). In the formation of waterꢀdispersed singleꢀ
H
CH
2
O
2
(30%), KMnO
4 2 4 2
, H SO , NaCH COOH (NaAc), and
layer MnO NSs, SDS plays as both the precursor of the
3
COOH (HAc) were purchased from Nanjing Chemical
2
reductant and a structureꢀdirecting agent. As illustrated in
Fig.S2, SDS undergoes partially hydrolysis to produce
dodecanol in acidic solution which was selfꢀassembled with
SDS into a lamellar structure. Subsequently, a redox reaction in
Reagent (Nanjing, China) CO., Ltd. Glucose was purchased
from Xilong Chemical. (Guangdong, China). Glucose oxidase
from Aspergillus niger (GOD,10KU) was purchased from
Aladdin and stored in a refrigerator at ꢀ20
sulfate (SDS) was purchased from SunShineBio company
Nanjing China). Streptozotocin (98%, Sigma, Chemical Co., St.
℃. Sodium dodecyl
situ occurs between dodecanol and KMnO to yield lauric acid
4
and MnO . Thus, well waterꢀsoluble MnO NSs were coꢀ
(
2
2
modified with lauric acid and SDS attributing to the
coordination interactions of carboxyl group and unsaturated Mn
atoms and hydrophobic effect. The synthesized MnO2 NSs
were purified by centrifugation and repeated wash with
ultrapure water and ethanol. The purified MnO2 NSs after
freezerꢀdry was readily suspended in ultrapure water to form a
colloidal suspension, which was then dried on corresponding
substrates overnight at room temperature for the morphology
characterizations of TEM, AFM. For other material
characterizations, such as FTꢀIR and ICPꢀOES measurements,
bulk nanosheets dried from a concentrated solution without
substrate were used.
Louis, MO, USA). Humulin 70/30 recombinant human insulin
mixed injection (300 u/3 mL, Lilly Suzhou Pharmaceutical Co,
China). Fructose, lactose, maltose was purchased from Aladdin.
Various amino acids were acquired from Sinopharm Chemical
Reagent CO., Ltd. Albumin human (HSA) was purchased from
SigmaꢀAldrich. All serum samples were collected from typeꢀ1
diabetes mellitus SD rat induction by streptozotocin. All other
chemicals were of analytical reagent grade and used without
further purification. Ultrapure Water (18.2Mꢁ·cm) was used
throughout.
Apparatus
All absorption spectra were measured on
a UVꢀ1800
Procedures for detection of glucose by using the presented
glucometer in trimodal signals: UV absorption spectroscopy
and MRS and fluorescence spectroscopy
spectrophotometer (Shimadzu, Japan) equipped with a plotter
unit and a quartz cell (1 cm × 1 cm), kinetic experiments and
scan spectrum. Fluorescence spectra measurement was
performed on a HitachiꢀFꢀ4600 spectrofluorometer (Tokyo,
Due to hydrophobic effect and hydrogen bond interactions,
GOD molecules would be spontaneously entrapped on the
Japan). T
1
ꢀ and T
2
ꢀweighted MRI images of a certain amount of
, and GODꢀ
surface of MnO
2
NSs via noncovalent approach. An ensemble
MnO NSs in different concentrations of H O
2
2 2
2
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ARTICLE
glucometer was facile obtained by directly mixing of certain
amounts of GOD and MnO NSs in aqueous solution standing
at ambient environment for five minutes. series of
considerable aliquot of the ensemble glucometer was added into
a set of 5 mL alibrated text tubes on demand of the designated
2
A
c
measurements, 1mL of 10 mM NaAcꢀHAc (pH 5.6) buffer
solution and a series of glucose solutions were added in turn.
The final mixtures were then diluted to volume with ultrapure
water, and mixed thoroughly. Prior to measurements, the
0
mixture solution was incubated in a water bath at 37 C for 30 Fig.2 A structural illumination of the synthetic MnO
2
NSs in aqueous solution (A),
2
the representative absorption spectra for free MnO NSs, and the proposed
min. Then, the mixture was taken out from the water bath and
glucometer (B) (inset: photograph for glucometer aqueous solution with Tyndall
scattering light).
allowed to cool to room temperature for
measurements.
5 min for
mixture was taken out from the water bath and allowed to cool
to room temperature for 5 min for spectroscopic measurements
at 374 nm.
Serum samples collection from type-1 diabetes mellitus rat
Streptozotocin (STZ)ꢀinduced diabetes mellitus offers a very
costꢀeffective and expeditious technique that can be extensively
State：
3
0
used in most strains of rodents. Ten adult male SD rats
weighed between 200 ꢀ 250 g were housed in wire mesh cages
at room temperature. Veterinary care was provided by local
laboratory animal house unit in Faculty of Medicine, China
Pharmaceutical University. All animals were handled according
to the guidelines established by institutional animal care and
use committees. The rats were divided into two groups (five for
one group). All rats were firstly injected intraperitoneally with a
single dose of 60 mg/kg of streptozotocin dissolved in sodium
citrate buffer (0.1 mol L , pH was adjusted to 4.5) at a
concentration of 10 mg/ml immediately before use. After 3
days, fasting blood samples were collected through the retroꢀ
orbital route using capillary tubes to assess glucose level by
using a commercially available glucometer. Animals showing
blood glucose higher than 16.7 mM were considered as diabetic
and two of them were chosen to use throughout for the study.
One of diabetic rats was as followings treated with a 3U of
insulin. Changes of serum glucose in different level of typeꢀ1
diabetes mellitus induction with streptozotocin coupled with
insulin treatment were monitored in a fixed time interval. All
serum samples were collected by remove of the clot of whole
blood samples under certification that were initially withdrawn
from SD rats through retroꢀorbital route using capillary tubes.
All serum samples were subjected to an appropriate dilution
before analysis and no other pretreatments were necessary.
We will conscientiously abide by the ethical principles of
animal welfare, accept the supervision and inspection of the
committee of China Pharmaceutical University at any time, and
voluntarily accept the punishment if any infringement.
Results and discussion
Design and characterization of the ensemble glucometer
ꢀ1
Benefited from ultimate structure and fascinating optomagnetic
3
1ꢀ34
properties of MnO
glucometer with
2
,
a
we herein presented an ensemble
sandwiched structure programmable
enginerred by selfꢀassembly of GOD and MnO
solution as shown in Fig. 1. Ultrathin MnO
2
NSs in aqueous
NSs with high
2
specific surface area appeared to act as benign hosts to trap and
support GOD and signalꢀtransduction moeity with targetꢀ
activiation. The designed glucometer gave a selfꢀindicating
approach of direct readout triple signals of UV/MR/FL
simultaneously triggered by an cascade enzymatic reaction. In
this study, homogenous dispersion of MnO
2
NSs coꢀ
functionalized with lauric acid molecules and SDS (Fig. 2A and
Fig. S2 in ESI) was readily obtained by a redox reaction
2
9
according to a modified literature. The asꢀobtained MnO
was characterized with a broad absorption band from 220 to
00 nm orignating from dꢀd transition of Mn atoms with a
2
NSs
7
3
considerable molar extinction coefficiency (ε374 nm = 9.0 × 10
M cm ) (Fig. 2B and Fig S3 in ESI). Compared to free
MnO NSs, the ensemble glucometer showed no obvious
ꢀ1
ꢀ1
34
Procedures for colorimetric assay by using the presented
glucometer for detection of serum glucose
2
changes in absorption profile, only a slight decrease of
absorbance beyond 450 nm propably due to better dispersion of
nanosheets in aqueous solution with aid of GOD. A typical low
and high magnification TEM mage of free GOD, as presented
in Fig. 3A, indicates a monodispersed similar spherical
morphology with a meaning size of 1.8 nm as well as a
meaning height approximately of 1.32 nm was identified by
respresentative AFM(Fig.3 BꢀD) greatly smaller than the size
of stretch GOD in water solution (Fig. S4A). The asꢀobtained
In view of simplicity and convenience, exploration of the
ensemble glucometer composed of certain amounts of GOD
and MnO NSs for daily blood glucose monitoring by only
2
using of absorption spectroscopy. To a series of 5 mL
calibrated test tubes a designated glucometer composed of
ꢀ1
1
.5
NaAcꢀHAc (pH 5.60) buffer solution,
appropriate for 5.0 L) or different amounts of standard
ꢀ
g mL GOD and 35
ꢀ
M MnO
2
NSs, 1 mL of 10 mM
a
serum sample
(
ꢀ
glucose solution were sequentially added. The mixture solution
was mixed thoroughly before addition of a new solution. Prior
2
MnO NSs display transparent lamellar morphologies with
lateral sizes estimated to be ~200 nm with a meaning height
approximately 0.95 nm between 0.83 nm and 1.16 nm expected
to spectroscopic measurement
incubated in a water bath at 37 C for 30 min. Then
，the final mixture solution was
0
，the
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-
Fig.4 Representative TEM images of the designated glucometer containing of 1.5 ꢀg mL
GOD and 35 ꢀM MnO
1
2
NSs upon addition of 20 ꢀM (A), 50 ꢀM (B) and 100 ꢀM (C)
glucose (Inset: photographs of the corresponding solutions with Tyndall scattering light,
smaller-sized nanoparticles etched by
reaction marked with white arrows).
2 2
H O -generated by glucose-GOD catalytic
2 2 2
MnO NSs was effective decompsed by H O generated from
glucoseꢀspecfic biocatalytic reaction described above. As seen
from Fig. 4AꢀC, glucoseꢀcascade induced decomposition of the
designated glucometer was further justified by TEM images,
indicating that MnO
2
NSs was gradually etched into smallerꢀ
sized nanofragments starting from nanosheets edge and further
2
+
15
soluble Mn ions with significant dispearance of its color and
Tydall scatterring light. Once MnO NSs are dissolved, the
2
Fig.3 Representative TEM images for free GOD (A) (Inset: HR-TEM image and diameter collective changes in absorbance, MRS and fluorescence of the
distribution GOD), MnO
Tapping-mode AFM 2D and 3D images for free GOD (B, D), free MnO
MnO NS-GOD hybrid as the glucometer (J, L) with corresponding height profiles (C,G,K)
of six samples marked with lower-case letters from a to f.
2
NSs (E) and MnO
2
NS-GOD hybrid as the glucometer (I),
presented glucometer would be observed. Taking in account of
temperature and pH effect on GOD activity and H
decomposition MnO NSs reaction, the optimization conditions
of pH 5.60, 37 and 30 minutes were recommended to be used
2
NSs (F, H) and
2
2
O ꢀ
2
2
℃
for detection of glucose throughout (Fig. S6 in ESI). As a
colorimetric assay (Fig. 5A, B), the absorbance of the ensemble
glucometer solution gradually decreased as the concentration of
glucose increased, even observed by a nakedꢀeye change in
2
for the thickness of singleꢀlayer MnO NSs oneꢀside and twoꢀ
side coꢀmodified with lauric acid and SDS molecules (Fig. S4B)
confirmed by TEM (Fig.3E) and AFM (Fig. 3F and H)
analysises. Notably, more ample wrinkles and folds were
observed for the ensemble glucometer with a clusterꢀlike
morphology (Fig. 3I, J) and a significant increase in meaning
color. The decreased absorbance (
glucose concentration from 1 M to 50
function of A = 0.00485[glucose] + 0.0188 (
limit of detecting of (LOD) (3s) of the present GODꢀMnO
based nanosesor was 0.1 M, whilst the precision of for 11
M glucose was 3.5% (relative standard
ꢁ
A) linearly increased with
ꢀ
ꢀ
M with a calibration
2
ꢁ
R
=0.992). The
NSs
height to 8.56 nm (Fig. 3K, L) refered to free GOD and MnO
2
2
NSs, respectively. The height of hierarchical glucometer
between 7.76 nm and 10.03 nm is responsible for 3ꢀfolds and 4ꢀ
folds of the sandwiched structure by a fullꢀpacked single
ꢀ
replicate detection 10
deviation, RSD).
ꢀ
molecular layer of GOD into double flakes of MnO
2
NSs. Thus,
2
Wherein manganese ions in original MnO NSs modified
a multiple sandwiched structure of the ensemble glucometer
was implied to be spontaneously formed from GOD and MnO
NSs via multiple noncovalent interactions as shown in Fig.1.
with long alkyl chain of SDS and lauric acid was shielded from
aqueous environment (Fig.2A) , making no contribution to the
2
2
+
protons’ longitudinal or transverse relaxation. Once Mn ions
released by decomposition of MnO exhibited greatly changes
in both T ꢀ and T weighted MR signals compared to original
Magnetic nanosensors underlying the princple
Self-indicating of glucose using the ensemble glucomter by
UV/FL/MR signals
2
1
2
4
, 7, 36
2
MnO NSs.
2
Differ remarkably from MnO NS based either single channel
of OFF/ON or activatable magnetic resonance signals have
been designed to identify and quantify a wide variety of target
or multimodal sensors as previous reported, a selfꢀindicating
approach was developed just by direct readout of intrinsic
signals of the individual units of the ensemble glucometer. The
presented glucometer was inspired by combination of unique
3
7ꢀ39
analytes.
1 2
The visualization changes in T ꢀ and T weighted
MR images for the presented glucometer was found in the
presence of different amount of glucose (Fig.5C). Plots of the
reactivity of MnO
2
(Fig.S5 in ESI) and enzymatic performances
corresponding T
difference versus gluocose conctrations displayed a similar
relationship From 10 M to 800 M (Fig.5D and E).
The intrintic UV fluorescence of GOD was significantly
suppressed by MnO NSs through energy transfer or both inner
filter efffect process (Fig.S7 in ESI).
1 2
ꢀ and T ꢀ weighted MR signals intensity
of GOD. Dꢀglucose as a substance was catalyzed specifically
by GOD into gluconic acid and H O described as equation. 1.
2 2
3
5
ꢀ
ꢀ
2
1
1, 40
The caged
fluorescence could be lighted up again owing to the gradual
4
| J. Name., 2012, 00, 1-3
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1
v
Km
1
1
ꢀ
ꢃ
ꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢅ3ꢆ
Vmax
ꢁ
S
ꢂ
Vmax
1
Fig. 6 (A) The absorption spectra of the presented glucometer containing of 1.5 ꢀg mL-
GOD-32 ꢀM MnO NSs in various concentration of glucose (B) Plots of changes at
absorbance at 374 nm (ꢁA374nm A-A against glucose concentrations (Inset:
2
=
0
)
Lineweaver-Burke plots of the GOD-glucose reaction.)
Because GOD can specifically catalyze the oxidation of glucose
to quantitative production of H
given enzymatic activity, the rate of H
with the glucose concentration. In view of H
decomposition of MnO NSs, the absorbance decrease actually
2
O
2, it is expected that for a
production increase
ꢀinduced
2
O
2
2
O
2
2
embodies the changes of GOD enzymatic activity. Seen from
Fig. 6, the absorbance changes increases with the increasing
glucose concentration up to 100
saturation. Thus, a 0.051 mM of
ꢀ
M, after which it reaches
K
m
was calculated according to
the equation describedꢀabove as shown in Fig. 6B. It is
exceptional 110 folds improvement of enzymatic activity over
Fig.5 (A) The absorption spectra of the ensemble glucometer at varied glucose free GOD with a
m
K of 5.85 mM which is greatly lower than
solutions in pH 5.6 of 10 mM HAc-NaAc buffer (Inset: photographs of the glucometer that of either covalent conjugation with gold NPs, Fe O NPs,
3
4
solution by addition of a series concentrations of glucose); (B) Plots of the decreased
absorbance as function of glucose concentration. (C) T -and T -weighted MR imaging of
the glucometer solutions containing of 5.0 ꢀg mL GOD-100 ꢀM MnO NSs at varied
concentrations of glucose; Plots of changes on T - (D) and T -(E) weighted MR signals
Mnꢀdoped ZnS QDs or noncovalent immobilized on
ZrO /chitosan, ZnO nanorods, graphene as far (Table S1 in
ESI). The enzymatic activity is generally acknowledged to be
1
2
-
1
2
2
1
2
intensity as a function of glucose concentrations. (F) Fluorescence spectra of free 20 ꢀg directly related with its second and tertiary structures which are
-
1
-1
mL GOD, the ensemble glucometer solutions containing of 20 ꢀg mL GOD-150 ꢀM substantial dependent on the interactions between enzymes and
MnO
2
NSs at varied concentrations of glucose, and (G) plot of corresponding ꢁF345 nm/F
0
19, 20, 42
asꢀimmobilized supports.
overall absorption profile of the presented glucometer was just
a superimposition of MnO NSs and GOD without new
absorption band and shift of band (Fig.S8 in ESI). Furthermore,
a zeta potential of initial MnO NSs with ꢀ24.5 mV changed to ꢀ
6.2 mV by addition of negative charged GOD in the pH of 5.6
FTꢀIR data showed that the
as a function of glucose concentrations.
2
decomposition of MnO
2
NSs by H
2
O
2
generated from glucoseꢀ
GOD biocatalytic reaction described above (Fig.5F). Plots of
the fluorescence intensity difference of glucometer (
versus gluocose concentrations gave a doseꢀdependent response
from 10.0 M to 100 M.
With respect to sensitivity and linearable response to
glucose, the colorimetric assay using the presented glucometer
was particularly favorable in point of care testing free of
requirements for either sophisticated instrument or much
2
0
ꢁF345nm/F )
2
buffer solutions (isoelectronic point of GOD being 4.2). The
FTꢀIR and zeta potential results suggested that a nonconvalent
interacation occurred to the process of selfꢀassembly of GOD
2
NSs via hydrogen bond and hydrophobic effect.
The conformational changes of GOD induced by MnO
were systematically investigated by CD spectroscopy, and
synchronous fluorescence spectroscopy and electrochemistry
test. CD spectroscopy results demonstrated that CD spectrum of
ꢀ
ꢀ
and MnO
2
NSs
1
7, 41
specific knowledge for luminescence and magnetism.
Mechanism on the enzymatic activity of GOD self-
assembled MnO NSs
free GOD has characteristic peaks at
which are retained in the native conformation. Whereas, GOD
immobilized by MnO NSs gave new peaks at 208 nm and 216
nm, respectively demonstrating that the precise enzymatic
conformation, including ꢀhelix, ꢀsheet, ꢀturn, and random
λ = 210 nm and 219 nm,
2
The enzymatic activity of immobilized enzymes gave an
extremely important effect of on the preformances of the
ensemble sensing system. To be our wellꢀknown that the
MichaelisꢀMenton parameters (
to evaluate enzyme activity. The parameters were quantitatively
2
α
β
β
m
K and Vmax) are generally used
coil, changes greatly by contrast to the free GOD (Fig. 7A and
Table S2 in ESI). The conformational changes implied a
4
2
estimated by use of the LinweaverꢀBurke plot as :
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ARTICLE
Journal Name
characteristic synchronous fluorescence originating from
tyrosine residue demonstrated transfer tyrosine residues to a
more hydrophobic environment accompanied with the partial
exposure of of FAD as a redox active center of GOD.
In view of reaction equilibrium theory, rectants in cycles
onꢀdemand between GODꢀglucose catalytic reaction and H
induced decompsition of MnO NSs undoubtedly promotes the
decomposition of MnO NSs (Equations 1 and 2) which is
2 2
O ꢀ
2
2
favorable for the whole reaction system essential relavant to
improvement of enzymatic activity. Last but not least that effect
2
+
of trace Mn ions released from MnO
activity and H ꢀinduced decomposition of MnO
with absorbance changes of MnO NSs. According to Equation
2
NSs both on GOD
2
O
2
2
was related
2
1
for glucoseꢀGOD catalytic oxidation, the activity of GOD
also could be directly monitored of pH value changes owing to
4
5
a production of gluconic acid. Cofactor effect of selfꢀ
2
+
generating of Mn ions on the improvement of enzymatic
activity of GOD was confirmed by a much faster and larger
change of pH of glucoseꢀGOD system at the identical initial pH
8
of 5.65 similar to DNAzyme (Fig. 7E). Furthermore, trace
2
+
Mn ions also gave an stimulation effect on H O ꢀinduced
2
2
decomposition of MnO2 NSs indicating by characteristic
absorbance evaluation with time, probably originating from
Fenton chemistry (Fig. 7F).
In a word, comprehensive enhancement enzymatic activity
of GOD with MnO2 NSs of the ensemble glucometer with
favorable configuration of GOD coupled with the partial
exposure of active center, onꢀdemand cyclic processes and selfꢀ
-
1
Fig.7 (A) CD spectra of 30 ꢀg mL free GOD (red line), 30 ꢀM MnO
their mixture (blue line), respectively. All samples were dissolved in HAc-NaAc (10 mM,
pH 5.6) (B) cyclic voltammetry of MnO NSs|GCE (black line) GOD|GCE (red line) and
GOD–MnO NSs|GCE (blue line) in N -saturated PBS 0.1 M with a pH 5.6 and scan rate
of 100 mV s . The synchronous fluorescence (ꢁλ = 15 nm) (C) and (ꢁλ = 60 nm) (D)
2
NSs (black line) and
4
6
2
2
2
-
1
-
1
spectra of 20 ꢀg mL GOD at varied concentration of MnO
function of MnO NSs concentration and Normalized fluorescence profiles of GOD
before and after addition of MnO
2 0
NSs (Inset: plots of F /F as a
2
2+
NSs);(E) Effect of trace Mn ions on the pH changes generating manganese (II) ions synergistic catalysis effects
of 1.5 ꢀg mL GOD-0.12 mM glucose system containing of 2.0 mM KCl from an identical including afforded a unusual enhancement of the enzymatic
2
-
1
2+
pH of 5.65 (F) Effect of Mn ions on 12 ꢀM H
MnO NSs.
2 2
O -induced decomposition of 35 ꢀM
activity of GOD of the ensemble glucometer.
2
Glucose-specificity and recycling response of the designed
favorable configuration of GOD with a higher affinity to glucometer
glucose, resulting to an improvement of enzymatic activity. The
The targetꢀspecific and recycle of the designated glucometer is
direct electrochemistry of GOD using cyclic voltammetry
technology, a pair of quasiꢀreversible redox peaks was only
of great significance for point of care diagnostic analysis. To
access the selectivity of the proposed ensemble glucometer
composed of GODꢀMnO2 NSs, we tested the influence of
conventional relevant metal ions, anions and other molecules in
observed at GODꢀMnO
with the formal peak potentials ꢀ0.354 V and peakꢀtoꢀpeak
separation ( ) 32 mV assigned to a charateristic electron
transfer of FAD (Fig. 7B) to electrode primary attributing to
2
NSs|GCE (glassy carbon electrode)
ꢁE
p
biological fluids on the detection of glucose (50 ꢀM).
Remarkably, as shown in Fig. 8, no obvious absorbance
changes were observed typical metal ions and anions, amino
acids and protein. Furthermore, those potential physiologically
substances showed negligible effects on the signal for glucose
sensing. Taking advantage of substanceꢀspecificity of GOD,
several glucose analogues, including maltose, lactose and
fructose gave little effect on glucose detection even at its
concentrations 6 times higher than that of glucose. Furthermore,
considering natural levels of glucose in human blood serum
4
3
partial exposure of enzyme active center. The conformational
changes were further be identified by fluorescence
4
4
spectroscopy.
The synchronous
fluorescence
of
GOD
was
characteristically collected by ꢁλ = 15 nm and ꢁλ = 60 nm,
assigned to aromatic tyrosine and tryptophan residues which
were important for the correct orientation of the substrate as
well as for the maximal velocity of glucose oxidation.
Concentration of MnO
2
NSs dependent fluorescence of GOD
(
4.4 to 6.6 mM), while the working range of the proposed
was well followed a SternꢀVolmer equation 4:
method is in the micromolar range with a LOD low to 0.1 M,
ꢀ
relative weak UV absorption signal of blood samples could be
largely eliminated only by facile dilution (Fig.S9 in ESI). Thus,
ꢄꢄꢄF0 F=1+Ksv MnO NSs
⁄
ꢁ
ꢂ
(4)ꢄ
2
Wherein a larger quenching constant (Ksv) was obtained for the proposed glucometer would work well with small amounts
tryptophan residue over tyrosine one that suggested interactions of serum samples, and physiologically relevant interferences as
between MnO NSs and GOD was closer to tryptophan residue well as matrix effect can be simply eliminated by direct dilution.
2
(Fig.7C and D). Additionally,
a
slight blueꢀshift of Notably, the recycle use of the ensemble glucometer is essential
6
| J. Name., 2012, 00, 1-3
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Jou Pr nl e aa ls oe fd Mo an to et r ai ad lj su s Ct hm ea mr g ii sn ts ry B
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ARTICLE
Fig. 9 Blood glucose measurements at a fixed time interval of two representative SD
rats of type 1 diabetes mellitus before and after treatment of insulin by using of the
presented glucometer (white column) and commercially available blood glucose meter
Fig.8 The designated glucometer based colorimetric system response towards various
metal cation ions and anions (A), various amino acids and protein (B). The white bars
represent the addition of an excess of metal cation ions and anions, various amino
acids and protein; The black bars represent the subsequent addition of 50 mM glucose
(
black column).
diabetic level again resulting from irreversible beta cell
+
2+
2+
2+
to the corresponding solution described-above (1 mM for K , Zn , Ca and Mg and
47
destruction (Fig. 9B).
3
+
other anions; 0.5 mM for Fe ) and traditional sugar analogues (C) Inset: the color
change with the corresponding solutions.) (D) The absorbance changes of the
presented glucometer containing of MnO
2
NSs with successively added in series and Conclusions
GOD in the presence of an identical glucose of 50 ꢀM.
In conclusion, we report that a versatile glucometer was
designed by selfꢀassembly of GOD and MnO
noncovalent interactions. Taking advantages of glucose oxidase
specificity and unique reactivity of MnO nanosheets, a selfꢀ
2
NSs via
for clinic diagnosis in view of costꢀeffective. To evaluate the
cycle use of the designed glucometer constructed by
noncovalent immobilization, a series of identical amount of
2
indicating approach for detection of glucose was realized by
direct readout of fluorescence/UV/MR signals free of
complicated operations. Comprehensive effects including
favorable configuration of GOD, onꢀdemand cyclic processes
and selfꢀgenerating manganese (II) ions synergistic catalysis on
the exceptional improvement of enzyme activity afforded the
presented glucometer with a high sensitive response to glucose.
Especially, a colorimetric assay by using the ensemble
glucometer with high sensitivity was preferentially available in
routine blood glucose monitoring available for type I diabetes
mellitus of rats. Meanwhile, the proposed multimodal
nanoprobe is readily generalized in principle for a number of
biomarkers for point of care diagnostic.
MnO
a fixed GOD of 0.15
Fig.8D, similar changes at characteristics absorbance at 374 nm
of the presented glucometer were observed after addition of
50 ꢀM glucose. The ensemble glucometer characterized with
2
NSs was successive added to a solution of containing of
ꢀ1
ꢀ
g mL in HAcꢀNaAc (1 mM, pH 5.6). In
targetꢀspecificity and recycle recognition allows it to accurate
quantitative detect of glucose in in vitro diagnosis.
Colorimetric assay for routine blood glucose monitoring by
using of the presented glucometer
To validate the applicative potential of the hybrid glucometer
for routine blood glucose monitoring by only utilize of
absorption spectroscopy with respect to simplicity and
convenience, we performed daily serum glucose assay of two
representative rats chosen from typeꢀ1 diabetes mellitus
induction by injected intraperitoneally with a dose of 60 mg/Kg
streptozotocin. All serum samples were collected by remove of
the clot of whole blood samples that were initially withdrawn
from SD rats through retroꢀorbital route using capillary tubes.
No additional pretreatments of the serum samples except simple
dilution were employed for glucose detection by using the
designated glucometer. Shown in Fig.9, a well agreement in the
results of serum glucose in various levels was obtained by two
methods describedꢀabove, displaying the accuracy of the
designated glucometer for glucose monitoring. A rat showing
continuous and stable blood glucose higher than 16.7 mM was
considered as diabetic (Fig. 9A). Insulinꢀtreated diabetics
during a 24ꢀhour period showed a significant decrease of blood
glucose to normal level while subsequent to an increase to
Acknowledgements
The authors are grateful for financial support from the National
Natural Science Foundation of China (Grant No. 21350161)
and the Natural Science Founding of Jiangsu Province (Grant
No. BK20130643), the Fundamental Research Funds for the
Central Universities (Grant No. PT2014YX0045), State Key
Laboratory of Medicinal Chemical Biology (Grant
No.20130404) and Key Laboratory of Drug Quality Control
and
Postdoctoral foundation of Jiangsu Province (Grant No.
402153C).
Pharcovigilance
(Grant
No.
DQCP2015MS06).
1
Notes and references
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Journal Name
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Table of content
It is the first time to present a glucose-activatable trimodal glucometer with an exceptional
enhanced enzymatic activity self-assembled from glucose oxidase and MnO Nanosheets for
2
diabetes monitoring in vitro.