The screening of metal ion inhibitors for glucose oxidase based on the peroxidase-like activity of nano-Fe3O4

Article abstract of DOI:10.1039/c7ra07081k

In this study, a colorimetric method is proposed based on the peroxidase-like activity of Fe₃O₄ magnetic nanoparticles for screening metal ion inhibitors for glucose oxidase activity. First, the glucose oxidase was typically used as a specific enzyme to catalyze the oxidation of β-d-glucose resulting in the generation of hydrogen peroxide. Next, having an inherent peroxidase-like activity, Fe₃O₄ magnetic nanoparticles were adopted as the catalyst. Then, the generated H₂O₂ was capable of participating in the oxidation of 3,3′,5,5′-tetramethylbenzidine to yield a blue colored product. Based on the above results, an in vitro screen model of metal ion inhibitors of glucose oxidase was thus established. Metal ions including Ca²⁺, Pb²⁺, Mn²⁺, Ag⁺, Al³⁺, Cu²⁺, Mg²⁺ and Zn²⁺ have been tested. Herein, towards the glucose oxidase activity, Ca²⁺, Pb²⁺, Mg²⁺ and Mn²⁺ showed no effect while Al³⁺ and Zn²⁺ displayed a slight activation, while of Ag⁺ and Cu²⁺ expressed a strong inhibition. The further detection of Ag⁺ and Cu²⁺ manifested that their IC₅₀ were 0.662 μmol L^-1 and 12.619 μmol L^-1, respectively. The entire detection process could be accomplished within 15 min. This assay is economical, time-saving and highly-effective with definitely significant reference for the screening of metal ions as glucose oxidase inhibitors.

Full text of DOI:10.1039/c7ra07081k

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The screening of metal ion inhibitors for glucose
oxidase based on the peroxidase-like activity of
nano-Fe3O4
Cite this: RSC Adv., 2017, 7, 47309
*
Yao-hui Wu, Lei Chu, Wen Liu, Lun Jiang, Xiao-yong Chen, Yong-hong Wang
*
and Yun-lin Zhao
In this study, a colorimetric method is proposed based on the peroxidase-like activity of Fe3O4 magnetic
nanoparticles for screening metal ion inhibitors for glucose oxidase activity. First, the glucose oxidase
was typically used as a specific enzyme to catalyze the oxidation of b-D-glucose resulting in the
generation of hydrogen peroxide. Next, having an inherent peroxidase-like activity, Fe3O4 magnetic
nanoparticles were adopted as the catalyst. Then, the generated H2O2 was capable of participating in the
oxidation of 3,30,5,50-tetramethylbenzidine to yield a blue colored product. Based on the above results,
an in vitro screen model of metal ion inhibitors of glucose oxidase was thus established. Metal ions
including Ca2+, Pb2+, Mn2+, Ag+, Al3+, Cu2+, Mg2+ and Zn2+ have been tested. Herein, towards the
glucose oxidase activity, Ca2+, Pb2+, Mg2+ and Mn2+ showed no effect while Al3+ and Zn2+ displayed
a slight activation, while of Ag+ and Cu2+ expressed a strong inhibition. The further detection of Ag+ and
Cu2+ manifested that their IC50 were 0.662 mmol Lꢀ1 and 12.619 mmol Lꢀ1, respectively. The entire
detection process could be accomplished within 15 min. This assay is economical, time-saving and
highly-effective with definitely significant reference for the screening of metal ions as glucose oxidase
inhibitors.
Received 26th June 2017
Accepted 13th September 2017
DOI: 10.1039/c7ra07081k
rsc.li/rsc-advances
been seriously threatening the health of human beings, hence
the detection of these metal ions is of increasingly widespread
1. Introduction
importance.
In terms of environment contamination, metal ions are now
ranked one of the most severe contaminants. Among them, lead
and mercury1 are the common heavy metals, which can cause
damage to the central nervous system. Lead poisoning can
cause a variety of toxic symptoms such as anemia, dizziness,
hallucination, headache, insomnia, irritability, weakness of
muscles and renal damage. Mercury poisoning causes chest
pain, dyspnoea and impairment of pulmonary and kidney
function. In addition, the familiar heavy metal ions, cadmium
and nickel, are certied as human carcinogens.2 Cadmium
accumulated in the human body can lead to renal dysfunction
and in some cases even cause death on high degree of accu-
mulation. High levels of exposure of nickel result in serious
lung and kidney problems, such as skin dermatitis, pulmonary
brosis and gastrointestinal distress. In addition, zinc and
copper, which play an important role in the process of growth
and development of organisms, bring about serious toxicolog-
ical problems such as cramps, convulsions and even death on
excessive ingestion.3 The pollution caused by metal ions has
Many methods have been applied to detect the presence of
metal ions, including high performance liquid chromatography
(HPLC),4 thin-layer chromatography (TLC),5 chemiluminescent
immunoassay (CLIA),6 surface enhanced Raman spectroscopy
(SERS),7 uorescent spectrometry,8 electrochemical method9
and enzyme-inhibition.10 Among the various methods used for
the detection of metal ions thus far, due to its selectivity, the
inhibition to enzyme activity offered an appealing choice for the
determination of metal ions.11 Hence, using enzyme-inhibition,
the analysis of metal ions attracted a remarkable interest from
researchers home and abroad. A variety of enzymes, such as
alkaline phosphatase, invertase, catalase, glucose oxidase (GOx)
and urease were applied for the detection of metal ions, of
which GOx and urease ranked among the most widely reported,
showing promise in many cases to be applied for real samples
based on the inhibition effect.12 In particular, initially discov-
ered in Aspergillus niger extracts13 and generally regarded to be
one of the most constantly used enzymes in the eld of
biotechnology, GOx acted as an analytical reagent used for the
study of inhibition. Guascito et al.14 used glucose oxidase to
successfully develop a screen-printed electrode based ampero-
metric biosensor for the inhibitive detection of heavy metal
ions. Rust et al.15 immobilized glucose oxidase on nitrogen-
Key Laboratory of Forestry Remote Sensing Based Big Data & Ecological Security for
Hunan Province, College of Life Science and Technology, Forestry Biotechnology
Hunan Key Laboratories, Central South University of Forestry and Technology,
410004, Changsha, China. E-mail: bionano@163.com
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doped carbon nanotubes (N-CNTs) to successfully develop an Jinsui Biological Technology Co., Ltd. (Shanghai, China). GOx
amperometric glucose biosensor for the determination of silver was purchased from Yuanye Biological Technology Co., Ltd.
ions. Ayenimo et al.16 proposed a method describing the (Shanghai, China). The above regents were analytical or
detection of Cu2+, Hg2+, Cd2+ and Pb2+ through inhibition of the biochemical grade and were used as received. The above solu-
response of an ultrathin polypyrrole–glucose oxidase (PPy–GOx) tions were prepared with deionized water. Male healthy urine
potentiometric biosensor.
samples were collected from the volunteers and provided by the
Although the abovementioned biosensor assays used for the Hospital of Central South University of Forestry and Tech-
inhibitive detection of metal ions were accurate, reproducible nology. All of the metal ion solutions were diluted with deion-
and highly specic, it depended on the use of expensive ized water or male healthy urine.
instruments. Compared with biosensor assays, colorimetric
assays have attracted considerable interest in metal ions
inhibitive detection because of their simplicity, rapid response
and cost-effectiveness. GOx could catalyze the oxidation of beta-
2.2 Instruments
HH-2 digital electric-heated thermostatic water bath (Shanghai,
China) was used for controlling the reaction temperature. Pure
D-glucose to D-gluconic acid and hydrogen peroxide (H2O2) with
water preparation system (Qingdao, China) was used to manu-
oxygen molecules as electron acceptors.17 Based on the reaction
facture the deionized water. A JB90-D electric mixer (Shanghai,
China) was applied to stir the reaction of synthesizing Fe3O4
mechanism of GOx, the glucose–GOx system was usually
studied by coupling with a peroxidase-catalyzed color system.
MNPs. A vortex mixer (Labnet International, Inc.) was adopted
Research and development of mimic enzyme have indicated
to mix the reactions. Scanning electron microscope (SEM,
that nanostructured materials as peroxidase mimetics display
Hitachi S-3400N, Japan) was applied to determine the
unparalleled advantages of stability and low-cost compared
morphology of the MNPs. Magnetic properties of Fe3O4 MNPs
with natural enzymes,18 of which magnetic nanoparticles
were tested using a vibrating sample magnetometer (VSM,
(MNPs) were the most widely studied enzyme mimics. Gao
America) and a magnet (NdFeB). The UV-vis absorption and
color changes were recorded with a Nanodrop 2000 UV-vis
spectrophotometer (Hong Kong, China) and a digital camera,
respectively.
et al.19 have made a surprising discovery that Fe3O4 MNPs
possessed intrinsic peroxidase-like activity. Wang et al.,20 based
on the peroxidase-like activity of magnetic mesoporous silica
nanoparticles (Fe3O4@MSN), developed a colorimetric method
for the measurement of H2O2 and glucose.
In this study, we developed a novel model for the screening 2.3 Preparation of stock solutions
of metal ion inhibitors of GOx by coupling the glucose–GOx
system with the Fe3O4 MNPs-catalyzed color system. As
FeSO4$7H2O and KOH were dissolved in deionized water with
a stock concentration of 0.025 mol Lꢀ1 and 0.1 mol Lꢀ1
,
a mimetic enzyme, it was difficult to deactivate the magnetic
nanoparticles, ensuring the stability of the test results.
Furthermore, the MNPs could be separated easily by a magnet
so that the substances in the solution could be detected
conveniently, thus simplifying the operation of the entire
detection process. This strategy not only provided a platform for
the screening of GOx metal ion inhibitors in a novel, simple and
sensitive manner, but also held a huge potential to be utilized in
biomolecular diagnosis and studied using other essential
enzymes.
respectively. The TMB was dissolved in ethanol with a stock
concentration of 5 mg mLꢀ1. Both CH3COOH and CH3COONa
were dissolved in deionized water with a stock concentration of
0.2 mol Lꢀ1 and used with appropriate proportions. The b-D-
glucose and all the metal ion salts of crystalline solid were
dissolved in deionized water with a stock concentration of
1 mol Lꢀ1, and used with appropriate dilution. The GOx was
dissolved in 0.2 mol Lꢀ1 acetic acid–sodium acetate buffer
solution (pH 4.0) with a stock concentration of 0.25 mg mLꢀ1
ꢁ
and placed at 4 C.
2. Experimental
2.4 Preparation and characterization of Fe3O4 MNPs
2.1 Materials
Mono-dispersed globose Fe3O4 MNPs were synthesized by a re-
Iron(II) sulfate heptahydrate (FeSO4$7H2O), sodium hydroxide ported sol–gel method.21 The preparation process consisted of
(NaOH), b-D-glucose (C6H12O6$H2O) and potassium hydroxide the following steps. First, 200 mL of 0.025 mol Lꢀ1 FeSO4
(KOH) were obtained from Xilong Chemical Reagent Co., Ltd. solution was added into a beaker. Subsequently, 120 mL of
(Shanghai, China). Hydrogen peroxide (H2O2, 30%), lead(II) 0.1 mol Lꢀ1 KOH solution was added. Under the condition that
nitrate (Pb(NO3)2) and silver nitrate (AgNO3) were acquired from the temperature of the water bath was maintained at 80 ꢁC, 300
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). mL of 5% H2O2 solution was rapidly added to the mixture with
Acetic acid (CH3COOH) and sodium acetate (CH3COONa) were vigorous stirring for 10 min. The resulting black particles were
provided by Damao Chemical Reagent Factory (Tianjin, China). magnetically separated and repeatedly washed by deionized
Calcium nitrate (Ca(NO3)2$4H2O), magnesium nitrate water and ethanol, in sequence. The Fe3O4 MNPs were
(Mg(NO3)2$6H2O), aluminum nitrate (Al(NO3)2$9H2O), cupric dispersed in 200 mL deionized water, and stored in a narrow-
nitrate (Cu(NO3)2$3H2O) and zinc nitrate (Zn(NO3)2$6H2O) were mouth bottle. The morphology and distribution of the
bought from Fengchuan Chemical Reagent Co., Ltd. (Tianjin, product were characterized by a scanning electron microscope
China). 3,305,50-Tetramethylbenzidine (TMB) was obtained from (SEM). The magnetic properties of the obtained nanoparticles
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were veried using a vibrating sample magnetometer (VSM) and In the screened metal ions detection, ve concentration gradi-
a magnet.
ents for each ion were prepared through diluting 2 mmol Lꢀ1
metal ion solution for 10, 102, 103, 104 and 105 times, separately;
then the IC50 of the metal ion inhibitors were calculated.
2.5 Feasibility experiment
A glucose–GOx system was established as a research system. For
the glucose–GOx system, 2 mL graduated EP tubes were used as
reaction carriers and the reaction volume was dened as 1 mL,
including 680 mL of 0.2 mol Lꢀ1 acetic acid–sodium acetate
buffer solution (pH 4.0), 20 mL of 0.25 mg mLꢀ1 GOx solution
and 300 mL of 0.1 mol Lꢀ1 glucose solution. Prior to the enzy-
matic reaction, acetic acid–sodium acetate buffer solution and
GOx solution were added into a 2 mL graduated EP tube and
2.8 Detection of metal ions in urine
The screened metal ions were added to the urine and diluted to
the same ve concentrations as in the previous experiment. The
experimental results were compared with the previous ones.
3. Results and discussion
3.1 Research strategy
ꢁ
preheated for 3 min at a constant temperature of 37 C in the
thermostatic water bath. Aer the completion of the enzymatic
reaction, 500 mL of 1 mol Lꢀ1 NaOH solution was added into the
reaction system for the termination of reaction. The H2O2
produced in the enzymatic reaction was to be detected.
A color system was established for the detection of the
enzymatic reaction produced H2O2. For the color system, 5 mL
graduated EP tubes were used as the reaction carriers and the
reaction volume was dened as 3 mL, including 2.1 mL of
0.2 mol Lꢀ1 acetic acid–sodium acetate buffer solution (pH 3.0),
200 mL Fe3O4 MNPs dispersion, 100 mL of 5 mg mLꢀ1 TMB
solution and 600 mL H2O2 solution from the terminated enzy-
matic reaction. Aer reacting for 10 min at room temperature
(25 ꢁC), the Fe3O4 MNPs were magnetically aggregated at the
bottom of the EP tube, and thus the supernatant was pipetted
for detection. The measurements were conducted with a scan
range from 400 nm to 800 nm.
It is generally known that GOx is an ideal enzyme which cata-
lyzes the oxidation of b-D-glucose to D-gluconic and H2O2 (eqn
(1)). Due to its peroxidase-like activity, Fe3O4 MNPs are able to
catalyze the reaction of 3,30,5,50-tetramethylbenzidine (TMB) in
the presence of H2O2 to produce a blue color solution (eqn (2)).
GOx
ƒƒƒƒƒƒƒ!
glucose þ O þ H O
gluconic acid þ H O
(1)
(2)
2
2
2
2
Fe3O4 MNPs
H O þ TMB
H O þ oxTMB
ƒƒƒƒƒƒƒƒƒƒ!
2
2
2
We devised a strategy for the screening of GOx metal ion
inhibitors based on the above information as shown in Fig. 1. A
glucose–GOx system was used as a research system. A color
system catalyzed by Fe3O4 MNPs was established for the
detection of H2O2 produced in GOx-catalyzed reaction. When
the color reaction was completed, Fe3O4 MNPs were magneti-
cally separated using a magnet and the absorbance of the blue
product was obtained using a spectrophotometer. The effect of
metal ions on GOx activity was investigated based on the change
of absorbance before and aer GOx was treated with metal ions.
2.6 Optimal conditions of enzymatic reaction
In order to understand the relationship between the enzymatic
reaction rate and time and obtain the reaction time at which the
enzymatic reaction rate was constant, the reactions based on
the established glucose–GOx system were carried out from 20 to
300 s and followed the same procedure of detection of H2O2
based on the established color system. For the glucose–GOx
system, the inuencing factors involving the concentration of
substrate, temperature and pH were optimized. To optimize the
substrate concentrations, the series glucose solutions from
0.003 to 0.3 mol Lꢀ1 were investigated. To optimize the reaction
temperature, the reaction solutions incubated in different
ꢁ
temperature water baths ranging from 25 to 70 C were inves-
tigated. To optimize the pH of the reaction solution, 0.2 mol Lꢀ1
acetic acid–sodium acetate buffer solutions with a pH range of
3.0 to 5.0 were investigated. The processes of detection of H2O2
were similar to that of the feasibility experiment.
2.7 Metal ion inhibitors screening
The screening of metal ion inhibitions for GOx was performed
based on the optimized enzyme reaction conditions. The effects
of different metal ions on the enzymatic activity were investi-
gated. Some ions including Ca2+, Pb2+, Mn2+, Ag+, Al3+, Cu2+,
Mg2+ and Zn2+ were applied in this experiment. The nal
concentration for each ion was 2 mmol Lꢀ1. The metal ions that
Fig. 1 Schematic illustration of the proposed method for the metal ion
possessed inhibitory effect were screened for further detection. inhibitors screening of glucose oxidase.
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Table 1 Saturation magnetization and coercive field determined at
300k
This was because the activity of GOx was reected by the
absorbance of the blue product. The effect of metal ions on GOx
activity was effectively interpreted by the different absorbance of
the reaction solutions. Upon treating GOx with metal ions, there
could be three possible different results—the ineffective situa-
tion, the inhibited situation and the activated situation. We
focussed on studying the situation of the inhibitory effect
exhibited by the metal ions.
Saturation magnetization Ms
Coercive eld
Hc (G)
T (K)
(emu gꢀ1
)
300
15.242
5.1474
properties obtained by SEM and VSM, we detected the H2O2
produced in the GOx-catalyzed reaction using the established
Fe3O4 MNPs-catalyzed color system whose results were shown
in Fig. 2c. We found that the Fe3O4 MNPs catalyzed the reaction
of TMB to produce a blue color, with maximum absorbance at
652 nm. This was consistent with the ndings of Gao et al.19
This indicated that the prepared Fe3O4 MNPs indeed exhibited
the intrinsic peroxidase-like activity. Hence, the color system
catalyzed by Fe3O4 MNPs was applied for studying the glucose–
GOx system. In other words, the feasibility of the experiment
was proven.
3.2 Characterization of Fe3O4 MNPs and feasibility analysis
To investigate the feasibility of the experiment, we rst prepared
Fe3O4 MNPs, and then characterized the morphological struc-
ture and magnetic property of the MNPs. The obtained black
Fe3O4 particles were further characterized using scanning
electron microscope (SEM) and the recorded image is shown in
Fig. 2a. It was observed that the features of the Fe3O4 particles
were homogeneously spherical shaped and the diameter of each
particle was about 10 nm. The response of Fe3O4 particles to the
magnetic force was simply tested by a magnet whose results are
shown in Fig. 2b. It could be noticed that the originally
dispersed black particles rapidly aggregated to the bottom
under the action of the magnet. In addition, Fig. 2c displays the
magnetization curve for Fe3O4 MNPs and the values of the
saturation magnetization (Ms) and coercive eld (Hc) deduced
from the measurements are presented in Table 1. It could be
easily observed that the magnetization of Fe3O4 MNPs increased
with increasing magnetic eld. When the applied magnetic eld
reached 20 000 kG, the magnetization of the sample saturated
and its Ms value obtained was 15.242 emu gꢀ1. It was
also observed that its Hc was very small, about 5.1474 G, indi-
cating that Fe3O4 nanoparticles exhibited typical super-
paramagnetism. Based on the nanometric size and magnetic
3.3 Optimal conditions of enzymatic reaction
In order to ensure that the GOx was the most suitable for
playing its catalytic role, we optimized the reaction time,
substrate concentration, pH and temperature.
A curve representing the process of GOx-catalyzed reaction
was obtained (Fig. 3a) by the reaction from 20 to 300 s. The
reaction solutions were measured using a spectrophotometer at
652 nm. From Fig. 3a, it could be observed that the lag phase of
the enzymatic reaction process is not clear. This could be
caused by the preheating of GOx solution before the reaction.
Compared with the lag phase, the linear phase and the
nonlinear phase were more evident. From 20 to 60 s, the curve
presented a good linear relationship. It was suggested that the
Fig. 2 (a) SEM image of the synthesized Fe3O4 particles. (b) Response Fig. 3 (a) GOx-catalyzed reaction process curve. The time points are
of Fe3O4 MNPs to a magnet (NdFeB). (1) Fe3O4 MNPs dispersion liquid. 20, 40, 60, 120, 180, 240 and 300 s. (b) The effect of concentrations of
(2) Status that Fe3O4 MNPs had been gravitated by the magnet. (c) The substrate. The concentrations of substrate are 0.003, 0.015, 0.03, 0.15
magnetization curve for Fe3O4 MNPs. (d) UV-vis absorption spectra and 0.3 mol Lꢀ1. (c) The effect of reaction pH. The pH values are 3.0,
(400–800 nm) and the related optical images of the nano-Fe3O4 3.5, 4.0, 4.5 and 5.0. (d) The effect of reaction temperatures. The
catalyzed color reaction.
temperatures are 25, 30, 35, 40, 45, 50, 60 and 70 ꢁC.
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rate of enzyme reaction was consistent in this period. And with
prolonged reaction time, the curve gradually tended to be at
due to several interferences such as inhibitory effect of the
product, decrease in the substrate concentration and reduction
of the enzyme activity. It could be learned from Fig. 3a that the
time for the initial rate of the enzymatic reaction was from 20 to
60 s. In order to set aside enough time for the operation, the
reaction time was conrmed as 1 min.
To investigate the effect of substrate concentration on the
rate of GOx-catalyzed reaction, we tested through setting up
a different substrate concentrations (0.003–0.3 mol Lꢀ1). From
0.003 to 0.03 mol Lꢀ1, the relationship between the reaction rate
and the substrate concentration was proportional (Fig. 3b). This
indicated that the enzymatic reaction was manifested as a rst-
order reaction. From 0.03 to 0.15 mol Lꢀ1, the increase in
enzyme reaction rate was not proportional to the concentration
(Fig. 3b). This showed that the enzymatic reaction was man-
ifested as a mixed-order reaction. When the substrate concen-
tration exceeded 0.15 mol Lꢀ1, the rate of enzymatic reaction
was independent of the substrate concentration (Fig. 3b). This
suggested that the enzymatic reaction was manifested as a zero-
order reaction. This occurred because the GOx in the solution
were completely saturated with the substrate so that there was
no excess enzyme in the solution. Although the substrate
concentration was increased, no more intermediates were
produced. It was clear that the enzymatic reaction reached its
maximum reaction rate (Vmax) at a substrate concentration of
0.15 mol Lꢀ1. Therefore, the optimum substrate concentration
Fig. 4 The screening of GOx metal ion inhibitors. (a) UV-vis absorption
spectra (652 nm) and the related optical images of control group (GOx
untreated by metal ions) and experimental groups (GOx treated by
metal ions). (b) Effects of metal ions (2 mM) on GOx activity. The metal
ions are Ca2+, Pb2+, Mn2+, Ag+, Al3+, Cu2+, Mg2+ and Zn2+
.
denaturation. Thus, the rate of enzymatic reaction rapidly
decreased. According to the above analysis, the optimum
was determined to be 0.15 mol Lꢀ1
.
ꢁ
temperature was 40 C. The optimum conditions for the GOx-
To investigate the effect of pH on the rate of GOx-catalyzed
reaction, we tested through setting up different pH ranging
from 3 to 5. The resultant curve obtained is shown in Fig. 3c,
which is a bell-shaped curve and revealed that GOx exhibited
the highest catalytic activity at pH 4.0. This was because the
dissociation state of GOx at pH 4.0 was most favorable for the
combination with the substrate, while other pH affected the
conformation of the enzyme active site that affected the binding
to the substrate. Thus, pH 4.0 was xed as the optimum pH.
To investigate the effect of temperature on the rate of GOx-
catalyzed reaction, we tested through setting up different
temperatures ranging from 25 to 70 ꢁC. As shown in Fig. 3d,
from 25 to 40 ꢁC, the rate of enzymatic reaction accelerated with
the increase in temperature. The reason was that the denatur-
ation of GOx had not yet been observed at the range below its
catalyzed enzymatic reaction are listed in Table 2.
3.4 Metal ion inhibitors screening
In order to examine the possibility of screening for GOx metal
ion inhibitors, we analyzed the effects of several metal ions on
the enzyme. The screening in the presence of metal ion inhib-
itors was investigated using the optimal enzymatic reaction
conditions obtained above. As shown in Fig. 4a, the measured
absorbance was signicantly lower than the control (GOx
untreated by metal ions) when GOx was treated by Ag+ and Cu2+;
when GOx was treated by Ca2+, Pb2+, Mn2+, Al3+, Mg2+ and Zn2+,
the measured absorbance was similar to that of the control. The
inuencing degree of metal ions on GOx could be calculated
from Fig. 4a and the results are displayed in Fig. 4b. It is clear
that except Ag+ and Cu2+, other metal ions had no signicant
inhibitory effect on GOx activity. This was consistent with the
previous reports.22,23 Besides, the metal ions except Ag+ and
Cu2+, Ca2+, Pb2+, Mg2+ and Mn2+ showed no effect on GOx
activity, while Al3+ and Zn2+ expressed a slight activation. Ag+
and Cu2+ could prevent the reduced form of the avin cofactor
ꢁ
optimum temperature. However, from 40 to 70 C, the rate of
enzymatic reaction reduced with the increase in temperature.
This was because GOx started to denaturate when the temper-
ature was higher than its optimum temperature. The effect that
the enzymatic reaction rate accelerated with increase in
temperature was counteracted by the inuence of GOx
Table 2 Optimal conditions of the GOx-catalyzed reaction
Reaction time (min)
1
Substrate concentration (mol Lꢀ1
0.15
)
pH
4.0
Temperature (ꢁC)
40
Optimum conditions
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(FADH2) from reducing the electron acceptor so that the GOx
activity was inhibited. This explanation is based on the study
reported by Wang et al.,22 showing that GOx inhibition by metal
ions was closely linked with the coordination of the reduced
form of the avin cofactor (FADH2) in the active site of GOx with
the metal ion.
3.5 The detection of metal ions
In order to further conrm our results, we used Ag+ and Cu2+ to
study their inhibitory effect. In the test, both Ag+ and Cu2+ were Fig. 6 The comparison of absorbance measured by standard samples
diluted 10 times, 102 times, 103 times, 104 times and 105 times
and urine samples.
on the basis of 2 mmol Lꢀ1. Then the inhibition efficiency
curves for Ag+ (Fig. 5a) and Cu2+ (Fig. 5b) were obtained and
urine sample were not much different, indicating that the system
established in our study could be applied to actual samples.
their IC50 were respectively measured as 0.662 mmol Lꢀ1 and
12.619 mmol Lꢀ1. The IC50 value for Ag+ was signicantly smaller
than that for Cu2+, indicating that Ag+ displayed a stronger
4. Conclusions
inhibitive effect compared with Cu2+. This is in agreement with
the literature results.22,23 The only difference was that the IC50
value for Ag+ and Cu2+ measured in our study were about 100
times lower than those obtained by Wang et al.22
A new type of in vitro screening pattern had been developed, based
on the peroxidase-like activity of nano-Fe3O4. Using the estab-
lished screening pattern, the metal ion inhibitors of GOx were
successfully screened and detected. Our detection method used
TMB as the color reagent and Fe3O4 MNPs which were not easily
deactivated compared to the natural peroxidase, hence it had
a good visual effect and stability in the test results. Furthermore,
the proposed assay whose entire detection process was completed
within 15 min is rapid and the detection limit measured could
reach the levels of 10ꢀ2 micromoles per liter. However, although
our system could recognize and determine a variety of different
single metal ion inhibitors, it could determine mixed metal ion
inhibitors. Even so, the new in vitro screening model established
in our study is efficient, accurate and repeatable. Referring to this
approach, a framework could be provided for the development of
many different screening patterns of enzyme inhibitors.
In order to verify whether our detection method could be
applied to a certain live sample, the standard addition method
was used to add the metal ion inhibitors to the urine and the
urine samples were measured by the same method as discussed
in an earlier section (2.7 Metal ion inhibitors screening). The
measured results were compared with the previous data (as
shown in Fig. 6). From Fig. 6, it could be seen that for the
absorbance, the measured value of the standard sample and the
Ethical statement
Male healthy urine samples were provided by the Hospital of
Central South University of Forestry and Technology. The
collected urine samples were not allowed to be used for
purposes other than those used in our project. Under this
condition, the Hospital agreed that the collected urine samples
could be used in our project. The content and process of the
study followed the international and national promulgated
ethical requirements for biomedical research. All study partic-
ipants provided informed consent.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Fig. 5 The detection of screened out metal ions. (a) The effect of
different concentrations of Ag+ on GOx activity, a series of concen-
trations of Ag+ ranging from 2 ꢂ 10ꢀ8 to 2 ꢂ 10ꢀ3 mol Lꢀ1. (b) The
effect of different concentrations of Cu2+ on GOx activity, a series of
This study is supported in part by the Research Foundation of
Education Bureau of Hunan Province, China (Grant No. 16A227)
Key Technology R&D Program of Hunan Province (2016TP1014,
2016TP2007).
concentrations of Cu2+ ranging from 2 ꢂ 10ꢀ8 to 2 ꢂ 10ꢀ3 mol Lꢀ1
.
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