Discriminative Detection of Glutathione in Cell Lysates Based on Oxidase-Like Activity of Magnetic Nanoporous Graphene

Article abstract of DOI:10.1021/acs.analchem.8b04779

As the most abundant intracellular biothiol, glutathione (GSH) plays a central role in many cellular functions and has been proved to be associated with numerous clinical diseases. Nevertheless, it is still a challenge to detect GSH over other mercaptoamino acids owing to their similar structures and activities. In this paper, magnetic nanoporous graphene (MNPG) nanocomposites were prepared for the first time through partial combustion of graphene oxide (GO) and ferric chloride. Due to the combination of porous graphene and magnetic nanoparticles, the MNPG nanocomposites exhibited large specific surface area, fast mass, and electron transport kinetics, resulting in remarkable oxidase mimic activity and easy separation. On the basis of the inhibition effect of GSH on the MNPG-catalyzed oxidation of thiamine, a novel and simple method for fluorescence determination of GSH was established. The sensor displayed a good linear response in the range of 0.2-20 μM toward GSH with a limit of detection of 0.05 μM. High sensitivity and selectivity facilitated its practical application for discriminative detection of GSH levels in PC12 cell lysates. The presented assay will be a simple and powerful tool to monitor intracellular GSH levels for biomedical diagnosis. Furthermore, the MNPG nanocomposites will provide insights to construct nanoporous graphene-based hybrids and push forward the advancement of porous graphene for wide applications.

Full text of DOI:10.1021/acs.analchem.8b04779

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Article
Discriminative Detection of Glutathione in Cell Lysates based
on Oxidase-Like Activity of Magnetic Nanoporous Graphene
Haijuan Zhang, Jia Chen, Yali Yang, Li Wang, Zhan Li, and Hongdeng Qiu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04779 • Publication Date (Web): 20 Mar 2019
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Analytical Chemistry
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Discriminative Detection of Glutathione in Cell Lysates based on
Oxidase-Like Activity of Magnetic Nanoporous Graphene
Haijuan Zhang‖, Jia Chen‖, Yali Yang, Li Wang, Zhan Li, and Hongdeng Qiu*
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CAS Key Laboratory of Chemistry of Northwestern Plant Resources/Key Laboratory for Natural Medicine of Gansu
Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China
*Corresponding Author: Prof. Hongdeng Qiu, Fax: +86931 4968019; Tel: +86 931 4968877; E-mail: hdqiu@licp.cas.cn.
ABSTRACT: As the most abundant intracellular biothiol, glutathione (GSH) plays a central role in many cellular functions and
has been proved to be associated with numerous clinical diseases. Nevertheless, it is still a challenge to detect GSH over other
mercaptoamino-acids owing to their similar structures and activities. In this paper, magnetic nanoporous graphene (MNPG)
nanocomposites were prepared for the first time through partial combustion of GO and ferric chloride. Due to combination of
porous graphene and magnetic nanoparticles, the MNPG nanocomposites exhibited large specific surface area, fast mass and
electron transport kinetics, resulting in remarkable mimic oxidase activity and easy separation. Based on the inhibition effect of
GSH on the MNPG-catalyzed oxidation of thiamine, a novel and simple method for fluorescence determination of GSH was
established. The sensor displayed a good linear response in the range of 0.2-20 μM towards GSH with a limit of detection of 0.05
μM. High sensitivity and selectivity facilitated its practical application for discriminative detection of GSH levels in PC12 cell
lysates. The presented assay will enable a simple and powerful tool to monitor intracellular GSH levels for biomedical diagnosis.
Furthermore, the MNPG nanocomposites will provide insights to construct nanoporous graphene based hybrids, and push forward
the advancement of porous graphene for wide applications.
Glutathione (GSH), a thiol-containing tripeptide (γ-Glu-
Cys-Gly), is the most abundant intracellular non-protein thiol
and plays a pivotal role in maintaining redox homeostasis,
combating oxidative stress, and defending against free radicals
and toxins.1,2 In particular, abnormal levels of cellular GSH
have been closely associated with numerous clinical diseases,
such as acquired immune deficiency syndrome (AIDS),
cancer, heart problems, and neurodegenerative diseases.3-5
Hence, the determination of cellular GSH level is critically
important to better understand the role of GSH in biological
systems, which is also of importance for accurate diagnosis of
disease.
synthesis, good stability, low cost, and design flexibility, in
comparison with those of natural enzymes.11-13 So far, efforts
have been made to explore nanozymes materials for GSH
detection, such as Fe3O4 nanoparticles,14 manganese oxides,15
graphene dots,16 Au nanoparticles,17 metal organic
frameworks,18 and so on. Nevertheless, poor diffusion and low
catalytic activity of those nanozymes restrict the applications
in biological media. In order to address these drawbacks, the
construction of enzyme mimics materials with outstanding
diffusion and high catalytic activity is urgently needed.
Two-dimensional (2D) nanoporous graphene (NPG) has
attracted vast interests recently due to their extraordinary
physiochemical properties such as unique porous structures,
large surface area and excellent electronic conductivity.19 The
unordinary features facilitate the widely applications of NPG
in electrochemical capacitors, field effect transistors (FETs),
sensors, separation science and molecular sieving.20-24
Moreover, the nanopores on the graphene sheets can
effectively impede aggregation of intersheets, accelerate mass
transfer and electron diffusion.25,26 Combining the advantages
from individual components, NPG-based hybrid materials may
possess synergistic effects, resulting in enhanced catalytic
To date, various analytical techniques have been established
for GSH detection, such as high performance liquid
chromatography (HPLC), mass spectrometry, electrochemistry,
and immunoiluminescence, etc.6-10 Though most of these
approaches exhibit high sensitivity, they suffer from non-
negligible intrinsic shortcomings such as expensive
instruments, time-consuming operation process, or
complicated synthetic procedures. Spectrometry has emerged
as the most convenient and promising tool for GSH detection
owing to its operational simplicity, inexpensive cost and high
sensitivity. However, discriminative detection of GSH over
cysteine (Cys) and homocysteine (Hcy) remains a tough task
because of their similar structures and reactivity. Therefore, it
is still highly desired to develop facile GSH sensing
approaches with high sensitivity and selectivity to meet the
biological and clinical requirements.
activity
of
NPG-based
nanocomposites.
Magnetic
nanoparticles (NPs) have received considerable attention on
account of their remarkable properties of large surface-to-
volume ratio and superparamagnetism. The integration of
NPG and magnetic NPs into magnetic nanoporous graphene
(MNPG) nanocomposites may produce new and/or enhanced
performance that cannot be realized by either component alone,
resulting in potential applications in many fields. Though the
assembly of magnetic NPs on graphene oxide has been
In recent years, nanomaterial-based enzyme mimics
(nanozymes) have been broadly investigated as new artificial
enzymes towing to their remarkable properties including easy
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Analytical Chemistry
reported,27,28 integrating the magnetic NPs and NPG
nanocomposite has rarely been reported to date, which is
probably ascribed to the limitation in synthetic approach and
the difficulty in fabrication of high quality NPG-based hybrids.
Page 2 of 9
was 25.4% as determined by inductively coupled plasmas
atomic emissive spectrometry (ICP-AES, IRIS Advantage
ER/S, TJA). High resolution mass spectrum (HRMS) of
reaction product was recorded on a MicroTof Q II (Bruker,
USA). Raman spectra were explored with a laser scanning
confocal micro-Raman spectrometer (LabRAM HR Evolution,
HORIBA, France), and the samples were scanned in an
extended range of 100 to 4000 cm-1. Fluorescence (FL) spectra
were measured using a LS-55 fluorescence spectrometer
(Perkin-Elmer, USA) with a 1 mL quartz cuvette (1 cm optical
path). The FL spectra of the solution were recorded in the
wavelength of 400-600 nm.
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In our recent work, we proposed a simple and rapid method
for the preparation of porous graphene by partial combustion
of graphene oxide imperfectly covered by hydrotalcite.29
Enlightened by this previous study, we suppose that different
kinds of NPG/metal oxide nanocomposites could be achieved
by changing the salt precursors. Herein, we for the first time
proposed a new and simple approach to fabricate MNPG
nanocomposites through combustion reaction of graphene
oxide (GO) and FeCl3. Expectedly, the as-prepared MNPG
nanocomposites exhibit outstanding catalytic activity and
could be applied for sensitive and discriminative
determination of GSH in cell lysates.
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Preparation of MNPG. The nanocomposite of NPG and
ferric oxide was prepared through combustion reaction. In
brief, 1.0 g FeCl3∙6H2O was mixed with a solution of GO (1.0
mL, 5.0 mg∙mL-1) under continuous sonication for 1 h. Then
the mixture was filtered through quantitative filter paper so
that most of GO and a certain amount of FeCl3 would be
trapped together on the filter paper. The filter paper covered
by GO and FeCl3 was dried in vacuum oven at 60 °C. After
the temperature of the muffle furnace reached 450 °C, the
covered filter paper was placed on a tin foil and put into the
muffle furnace with tongs and ignited at 450 °C for 2 min
(timed with a stopwatch). Then the sheets on the tin foil was
taken out with tongs carefully and naturally cooled to room
temperature. Finally, the collected sheets were washed and
centrifuged with ultrapure water for 10 times to remove
unreacted FeCl3 and other residual impurities (until the
supernatant becomes colorless). At last, the product was
washed with ethanol and dried at 60 °C overnight. The
resultant composites were named as MNPG. Pure NPG can be
easily obtained by washing MNPG with diluted hydrochloric
acid.29
EXPERIMENTAL SECTION
Materials and Chemicals. Graphite powder and H2O2 were
obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO,
USA). Quantitative filter paper (12.5 cm) was bought from
Sinopharm Chemical Reagent Co. Ltd., China. GO was
prepared from graphite powder by a well-known Hummer's
method with some modifications.30 FeCl3∙6H2O, HCl, H2SO4,
NaNO3, KMnO4 and vitamin B1 (also named thiamine, TH)
were purchased from Aladdin Chemistry Co. Ltd. (Shanghai,
China). L-Cysteine (L-Cys), L-lysine (L-Lys), L-arginine (L-
Arg), L-ascorbic acid (L-AA), L-histidine (L-His), L-
methionine (L-Met), L-tyrosine (L-Tyr), L-tryptophan (L-Trp),
L-phenylalanine (L-Phe), homocysteine (Hcy), L-isoleucine
(L-Ile), L-threonine (L-Thr), L-alanine (L-Ala), L-serine (L-
Ser), L-valine (L-Val), L-glycine (L-Gly), glucose, catechol,
nicotinamide adenine dinucleotide phosphate (NADPH),
nicotinamide adenine dinucleotide (NADH) and glutathione
(GSH) were bought from Sangon Biological Engineering
Technology & Services Co., Ltd (Shanghai, China). Dialysis
membrane with a molecular weight cut off of 8000-14000
g∙mol−1 was purchased from Beijing Chemical Reagent Co.,
Ltd (Beijing, China). PC12 cells (neuron-like rat
pheochromocytoma cell line) were obtained from Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy
of Sciences. Dulbecco's Modified Eagle Medium (DMEM)
was purchased from Sigma-Aldrich (St. Louis, USA). All
reagents were of analytical grade and used as received without
any further purification. All aqueous solutions were prepared
with ultrapure water of resistivity not less than 18.2 MH cm
(Milli-Q plus 185 equip, USA).
Preparation of PC12 cell lysates samples. PC12 cells
were cultured in DMEM medium (10% FBS, 2 mM glutamine,
and 100 units∙mL-1 penicillin/streptomycin). To prepare PC12
cell lysates samples, the cells were harvested and resuspended
using KPE buffer (0.1% TritonX-100 and 0.6% sulfosalicyclic
acid in 0.1 M potassium phosphate buffer with 5 mM EDTA,
pH 7.5). Then the suspension was sonicated for 2-3 min,
followed by centrifugation at 3000 g for 4 min at 4 °C. The
obtained supernatant was ready for GSH assay. The first panel
was served as a vehicle control. For comparison, the other two
panels were pretreated with 6-hydroxydopamine and
thioctamide, respectively. Other experimental procedures were
the same as above-mentioned.
Detection of GSH in cell lysates samples. The
determination of GSH in the PC12 cell lysates was carried out
as follows: 710 μL of phosphate buffer solution (10 mM,
pH=12.0), 150 μL of MNPG nanocomposite (1 mg∙mL−1), and
40 μL of 20 mM TH were mixed with 100 μL varied
concentrations of GSH (with final concentrations of 0, 0.2, 0.5,
2, 4, 10, 20, 40 and 60 μM). Subsequently, the mixtures were
vibrated at 40 ºC for 5 min to allow the oxidative reaction of
TH. The generated fluorescence intensity at 445 nm was
recorded immediately. The concentrations of GSH in the PC12
cell lysates samples were estimated by using the standard
addition method. All the experiments were repeated for three
times.
Apparatus and Characterizations. Transmission electron
microscopy (TEM) and energy dispersive X-ray spectrum
(EDX) were performed with a Tecnai G2 TF20 transmission
electron microscope (FEI, USA). Fourier transform infrared
spectrum (FT-IR) was conducted on an IFS 120HR Fourier
transform infrared spectrometer (Bruker, Germany). X-Ray
diffraction (XRD) patterns were observed by means of X ′
Pert PRO X-ray diffractometer (PANalytical, Netherlands). X-
ray photoelectron spectroscopy (XPS) analyses were recorded
on an ESCALAB 250Xi spectrometer (ThermoFisher
Scientific, USA). The magnetic property of MNPG
nanocomposites was measured with a vibrating sample
magnetometer (VSM, Lakeshore Cryotronics 730, USA) at
room temperature. The content of Fe in MNPG nanocomposite
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Figure 1. Schematic illustration for the combustive synthesis processes of MNPG and NPG (A) and TEM images of GO (B), MNPG nanocomposite (C)
and NPG (D). Insets are the size distribution of the magnetic NPs (inset of C) and the nanopores (inset of D), which are calculated from 50 individual
particles and nanopores, respectively.
The crystallinity of pure NPG and MNPG nanocomposites
was investigated by X-ray diffractometer, as presented in
RESULTS AND DISCUSSION
Characterizations of MNPG. The process for preparing
MNPG from GO and ferric chloride under combustive
treatment was schemed in Figure 1A. Firstly, GO was added
to a high concentration solution of FeCl3. After sonicated to
disperse, the mixture was filtered through quantitative filter
paper, then a certain amount of GO and FeCl3 was trapped on
the filter paper. During the heating and drying process, the
Fe3+ ions were hydrolyzed to form Fe(OH)3,31 leading to the
incomplete coverage of Fe(OH)3 layer on the surface of GO.
By igniting, thermally stressed Fe(OH)3 would decompose
into α-Fe2O3, which could be served as template and
precursors.31,32 During the process of carbothermal reduction,
GO served as the reducing agent and carbon source (CGO). At a
high reaction temperature, the bare GO in uncovered region
would be burned, while the α-Fe2O3 were partially reduced to
Fe3O4, resulting in the resultant products.33 The TEM was
performed to characterize the morphological and structural
features of the samples. Figure 1B and 1C showed the TEM
images of GO and the resultant MNPG nanocomposite after
combustive treatment, respectively. As can be seen, numerous
nanoparticles were uniformly distributed on the graphene
sheet ( ≈ 6.5 nm). The EDX spectrum indicated that the
nanoparticles were composed of iron oxide (Figure S1).
Along with these nanoparticles, some nanopores also could be
observed on the surface of graphene with an average pore size
of ≈ 3.5 nm, giving clear evidence for the successful
preparation of MNPG nanocomposites. After washing out the
iron oxide particles with dilute hydrochloric acid, the porous
structure of NPG could be clearly observed (Figure 1D),
further proving the existence of nanopores in the composites.
Figure 2A. Compared with the XRD pattern of GO (Figure
S2A), the (001) crystal planes at 10.9º of GO nanosheet
disappeared, while the (002) crystal planes at 21.2º were found
in NPG and MNPG. The broad diffraction peak at 21.2º was
indexed to the irregular stacking of the NPG sheets. The
diffraction peak of iron oxide phases could also be identified
by XRD. The strong and sharp peaks appeared within 0-80°
indicated the existence of α-Fe2O3 and Fe3O4 crystalline
phases. The peaks and corresponding indexed planes appeared
at 30.04° (220), 35.44° (311), 43.03° (400), 54.03° (422),
56.95° (511), and 62.89° (440) matched well with magnetite
(JCPDS file No. 75-0033), which confirmed the existence of
Fe3O4.34 The peaks and corresponding indexed planes
appeared at 24.08° (012), 33.07° (104), 35.44° (110), 40.78°
(113), 49.38° (024), 54.03° (116), 62.48° (214), and 64.01°
(300) matched hematite (α-Fe2O3) (JCPDS file No. 33-0664).35
To further verify the existence of α-Fe2O3 and Fe3O4, magnetic
hysteresis loop of the composites was recorded by a VSM with
an applied field sweeping from -20 to 20 kOe (Figure 2B).
The magnetic hysteresis curve showed the reversible behavior
and nonlinear between the applied magnetic field and the
magnetization. With the increasing of applied magnetic field,
the magnetization tended to be saturated with an Ms value of
3.6 emu∙g-1, which facilitated the magnetic separation in
practical manipulation. On the basis of the XRD and VSM
results, we know that the nanoparticle features of this hybrid
material were α-Fe2O3 and Fe3O4.
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Figure 3. The wide-survey XPS spectrum (A) and high-resolution C1s (B),
O1s (C) and Fe2p (D) XPS spectrum of MNPG.
Figure 2. (A) XRD patterns of the resultant NPG and MNPG, (B)
magnetic hysteresis loops of MNPG, (C) Raman spectra and (D) FT-IR
spectra of the as-synthesized NPG and MNPG.
in Figure 3D, the high resolution spectrum of Fe 2p had
character istic Fe 2p3/2 and Fe 2p1/2 doublets of iron oxide
electrons. The Fe 2p peaks fitted well with the 2p sub shells of
Fe3+ and Fe2+, confirming the existence of Fe2+ and Fe3+ in the
nanocomposites.35 The peaks at 712.0 and 725.4 eV
Raman spectroscopy was also applied for further
characterization of the nanocomposite. The Raman spectrum
of GO revealed a D bond at 1361 cm−1, a G bond at 1602 cm−1
and a 2D/G’ bond at about 2720 cm-1 (Figure S2B). The G
band is ascribed to an E2g mode of graphite related to the
vibration of sp2 bonded carbon atoms, the D band peak is
associated with the structural defect or partially disordered
graphite domains, whereas the 2D/G’ bond is a second-order
two-phonon mode.36 After the combustive treatment, the
intensity ratio (ID/IG) decreased from 1.04 for pristine GO to
0.93 for NPG (Figure 2C). The decreasing D peak of Raman
shift indicated that high crystalline porous graphene could be
obtained after combustive treatment.37 The removal of oxygen
defects would render a much higher content of sp2 carbon,
which was indicated by a significantly increased G band peak.
Decreasing of ID/IG ratio indicated that the combustive of GO
could effectively remove the oxygen defects and partially
rebuild the sp2 network of graphene.38
corresponded to the Fe3+ in α-Fe2O3.44 The peaks at 710.6 and
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723.8 eV were related to the Fe2+ in Fe3O4.
XPS
measurements further proved the formation of α-Fe2O3 and
Fe3O4, which was in good agreement with the XRD results.
The relative atomic ratio of Fe3+/Fe2+ (γ) could be calculated
according to the following equation32:
Ekj
ni Ii

nj I j Eki
where ni and nj represent the number of surface atoms, Ii and
Ij represent the peak areas, and Ekj and Eki represent the
photoelectron kinetic energies. According to the peak areas of
Fe2+ and Fe3+ (Figure 3D), γ was estimated to be 2.24.
Furthermore, the total content of Fe among the nanocomposite
was determined to be 25.4% by ICP-AES. Accordingly, the
mass fraction of α-Fe2O3 and Fe3O4 in MNPG were 14.0% and
32.3%, respectively, with a ratio of 3:5. After washing out the
iron oxide with dilute HCl, pure NPG could be obtained,
accompanied by the disappearance of Fe 2p peaks (Figure S3).
Both XRD and XPS confirmed the successful preparation of
NPG and MNPG nanocomposites.
Figure 2D showed the FT-IR spectra of NPG and MNPG.
The peak at 3200-3500 cm-1 was from stretching vibrations of
O-H. The band at 2938 cm-1 was assigned to the bending
vibration of C-H. The absorption bands at 1575 and 1715 cm−1
were attributed to the stretching vibrations of C=C in benzene
ring and C=O of carboxylic acid, respectively, which were
corresponding to the graphene structure. The bands at 490 and
520 cm-1 were from C-O-Fe and Fe-O stretching vibrations,
confirming the formation of magnetic porous graphene
composites.
Mimick oxidase application of MNPG for detection of
GSH. It have been reported that Fe3O4 and GO exhibit
properties mimicking those of enzymes.46,47 These findings
motivate our curiosity to investigate the mimic enzyme
activity of MNPG because of the combination of Fe3O4 and
NPG. TH, a non-fluorescent compound, can be easily
converted to strong fluorescent thiochrome (TC) in basic
media by appropriate oxidants.48 Thus TH can also act as an
attractive fluorescent substrate for oxidative detection. In the
present work, the as-prepared nanocomposite could catalyze
the oxidation of TH, resulting in the generation of
fluorescence (the reaction scheme was given in Figure 4A).
Figure S4A depicted the FL response of the sensing system by
using different materials as catalysts. The system containing
only TH exhibited almost none FL intensity. After Fe3O4 or
NPG was added, the FL intensity of the solution was enhanced
Surface sensitive XPS reveals more information with regard
to the surface composition and the chemical states of elements
in MNPG nanocomposites. The full range XPS (Figure 3A)
clearly showed three peaks at 285.9, 532.0, and 710.0 eV,
which were attributed to C 1s, O 1s and Fe 2p, respectively.39
In C 1s spectrum (Figure 3B), the fitted peaks at 284.6, 285.0,
286.4, and 288.7 eV were assigned to C=C (sp3), C-C (sp3), C-
O (sp2), and O-C=O (sp2) groups, respectively.40 In O 1s
spectrum (Figure 3C), the peaks at 532.5 and 533.6 eV were
assigned to C=O and C-O bonds, respectively.41 The peak
about 530.3 eV was assigned to the oxygen in Fe-O,42 and the
peak located at 531.6 eV should be caused by the bond of Fe-
O-C formed between Fe3O4 and graphene.43 As shown
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for the substrate. As illustrated in Figure S6, the apparent
values of Km and Vmax were 120.8 μM and 212.8 nM s−1,
respectively. The Km value of MNPG with TH is much lower
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than that of the natural horseradish peroxidase (HRP, Km
=
434 μM), Acr+-Mes (129 μM) and fluorescein (Km =158 μM),
illustrating the higher oxidase-like activity of MNPG
nanocomposite. 52-54
Based on the above excellent properties of the MNPG
nanocomposite, a novel and simple sensing approach was
fabricated for GSH detection. Figure 4B depicted the FL
spectra of TC after the addition of varied concentrations of
GSH. Initially, the non-fluorescent TH was oxidized to TC
under the optimum conditions, resulting in high fluorescence.
After adding varied concentrations of GSH, the FL intensity at
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Figure 4. (A) Schematic illustration of the MNPG-based fluorescent assay
for GSH detection. (B) Fluorescence emission spectra of TH upon the
addition of various concentrations of GSH (from top to bottom, 0, 0.2, 0.5,
2, 4, 10, 20, 40, and 60 µM). (C) The linear calibration curve of GSH with
X-axis representing different concentrations of GSH ranging from 0.2-20
μM and Y-axis representing their corresponding fluorescence intensity at
445 nm. (D) Fluorescence response of the system toward GSH (20 μM)
and other interferents with the concentrations of 200 μM (3-18) and 2 μM
(19-22): 1, control; 2, GSH, 3, L-Trp, 4, L-His, 5, L-Met, 6, L-Arg, 7, L-
Lys, 8, L-Tyr, 9, L-Cys, 10, Hcy, 11, L-Phe, 12, L-Ile, 13, L-Thr, 14, L-
Ala, 15, L-Ser, 16, L-Val, 17, L-Gly, 18, glucose, 19, L-AA, 20, catechol,
21, NADPH, 22, NADH. Error bars represent the standard deviation of
three repeated measurements.
445 nm gradually decreased. As it is known that GSH could be
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oxidized into glutathione disulfide (GSSG),
which could
also be verified in Figure S7. The competing oxidation of
GSH and TH restrained the oxidation of TH, leading to the
decrease of fluorescent after the introduction of GSH. The
gradual decrease of FL intensity revealed the sensitivity of the
system towards GSH concentration. As shown in Figure 4C,
there displayed a good linearity between the fluorescent of TC
versus the concentration of GSH ranging from 0.2 to 20 µM.
The limit of detection (LOD, calculated by 3σ/S, where σ
represents the standard deviation of blank signal, and S
represents the slope of the calibration curve) was 0.05 µM,
which was comparable with or even better than other
to a certain extent, while significant enhancement was
obtained after the addition of MNPG nanocomposite, implying
the higher oxidase-like activity of MNPG nanocomposite
compared to that of Fe3O4 and NPG. The high catalytic
activity of MNPG may be contributed to the following factors:
(1) the 3D architectures of NPG could provide high surface
area as well as fast mass and electronic transfer kinetics
because of the combination of the porous structures and
excellent intrinsic character of graphene;25-26 (2) Fe3O4 could
reduce the aggregation of graphene layers;49 (3) synergetic
effects between NPG and Fe3O4 in nanocomposite. On one
hand, the electrons involved in the carbon network of NPG
could donate electrons to reduce O2 via electron transfer. The
use of NPG “support” could provide abundant active sites and
greatly improve the stability and dispersion of Fe3O4 to allow
for better distribution.50 On the other hand, the Fe3O4 on the
graphene sheets could also serve as electron carrier during
electron transfer.51 Moreover, Fe3O4 could effectively impede
aggregation of graphene intersheets, accelerate mass transfer
and electron diffusion. Collectively, these results may in turn
facilitate significantly enhanced oxidase-like activities of the
MNPG nanocomposite.
fluorescent or mimic enzymes-based methods (Table S1).4, 15-
17, 53, 56-63
The intracellular complexity possesses a great challenge for
the detection of GSH. Taking into account that the
mercaptoamino acids coexist in biological fluids, it is more
difficult to discriminate GSH from Cys/Hcy. Thus the
influence of a series of biologically relevant amino acids
(especially L-Cys and Hcy) and some other reductive species
(glucose, L-AA, catechol, NADPH and NADH) were
evaluated. As shown in Figure 4D, only GSH induced
effective fluorescence reduction under the optimal conditions.
In stark contrast, neither L-Cys nor Hcy displayed
fluorescence interference at 445 nm. As GSH is the most
abundant intracellular reductive small molecule, its level is
much higher than the other reductive species.16 To better
simulate the intracellular condition, a concentration of 2 μM
was utilized for other reductive species. In this case, Glucose,
L-AA, catechol, NADPH and NADH were also negative to the
probe. Collectively, the proposed method possessed high
selectivity for GSH and could address the biological
requirements.
The BET surface area and average pore diameter of MNPG
(419 m2/g, 3.4 nm) were lower than that of NPG (482 m2/g,
3.5 nm), as shown in Figure S5. This could be due to the
filling or blocking of the surface and pores in NPG by α-Fe2O3
and Fe3O4 nanoparticles, resulting in a lower surface area and
pore diameter.
In virtue of the analytical advantages, the MNPG-based
sensor was also utilized for GSH detection in complicated
biological samples. As is reported, GSH is the most abundant
intracellular antioxidant for many mammalian. Besides, more
than 90% of the intracellular thiol molecule is GSH in most
cell samples. Here, PC12 cell was chosen as the model cancer
cell. Thioctamide was utilized as the stimulant agent in order
to induce GSH generation from cancer cells, and 6-
hydroxydopamine was used to reduce GSH generation. As
displayed in Table 1, a standard addition method was used by
spiking different concentrations of GSH into PC12 cell lysates.
The GSH level in the control cell lysates sample was
determined to be 0.40 mM, which was close to the normal
concentration of GSH in cells (0.5-10 mM).16 In order to
Some important factors affecting the fluorescence spectra of
the system were investigated, including the effect of TH and
MNPG concentration, pH values, reaction time and
temperature (Figure S4B-4F). Subsequently, oxidase-
mimicking activity of the MNPG nanocomposite was
investigated according to steady-state kinetic analysis. The
apparent kinetic parameters were calculated according to the
function: υ = Vmax × [S]/(Km + [S]), where υ represents the
initial velocity, Vmax represents the maximum reaction velocity,
[S] represents the substrate concentration, and Km represents
the Michaelis constant that reflects the affinity of the enzyme
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Table 1. Determination of GSH in PC12 cell lysates samples.
2
3
Samples
Detected (µM)
Added (µM)
Found (µM)
Recovery (%)
RSD (%)
94.0
106.0
103.5
92.0
6.3
1.9
2.8
5.2
3.2
3.6
4.8
5.0
10.0
20.0
5
404.0
409.9
420.0
336.0
340.8
351.7
456.3
4
5
6
7
8
9
Normal PC12 cell
lysates
399.3
Pretreated with 6-
hydroxydopamine
331.4
451.1
94.0
10
101.5
104.0
20
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
Pretreated with
thoctamide
95.0
97.0
3.7
5.4
10
20
460.6
470.5
confirm the feasibility of the proposed method, we carried out
other experiments by pretreating the PC12 cells with
thioctamide and 6- hydroxydopamine, respectively, to change
the GSH levels in cell lysates. As expected, the concentration
of GSH in cell lysates pretreated with thioctamide increased to
0.45 mM, while the one pretreated with 6-hydroxydopamine
decreased to 0.33 mM. These results together suggested that
the proposed method was suitable for detecting GSH levels in
cell lysates. Furthermore, the recoveries of given amount of
GSH in cell lysates samples were ranged from 92.0% to
106.0%, and the RSDs were ranged from 1.9% to 6.3%. The
satisfactory recoveries and RSDs definitely implied high
accuracy and reliability of the established method for the
detection of GSH in biological samples.
AUTHOR INFORMATION
Author Contributions
‖These two authors contributed equally to this work.
Notes
There are no conflicts of interest to declare.
ACKNOWLEDGMENT
The authors gratefully acknowledge the financial support
provided by the National Natural Science Foundation of China
(21822407, 21675164), the Natural Science Foundation of Gansu
Province (17JR5RA311, 18JR3RA387) and CAS “Light of West
China” Program.
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CONCLUSIONS
In summary, for the first time, a simple and rapid method to
fabricate MNPG nanocomposites was developed by partial
combustion of GO and ferric chloride. Combining the
advantages from both nanoporous graphene and magnetic
NPs, the MNPG nanocomposites possess high specific surface
area as well as fast mass and electron transport kinetics,
resulting in remarkable catalytic activity and easy separation.
On the basis of their mimic oxidase activity, the MNPG
nanocomposites have been successfully applied for
discriminative detection of GSH levels in PC12 cell lysates
with high sensitivity and selectivity. More notably, our
strategy will open new avenues for the fabrication of
nanoporous graphene based hybrids, but also inspire more
brilliant work in the future, and pave the way for the
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
EDX of the MNPG nanocomposite, XRD pattern and
raman spectrum of GO, survey XPS and high-resolution
C1s, O1s spectrum of NPG, nitrogen sorption isotherms
and pore size distribution curves of NPG and MNPG,
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spectrum of the reaction solution and comparison of
analytical performance with other methods.
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