Functionalized graphene oxide as a nanocarrier in a multienzyme labeling amplification strategy for ultrasensitive electrochemical immunoassay of phosphorylated p53 (S392).
ABSTRACT P53 phosphorylation plays an important role in many biological processes and might be used as a potential biomarker in clinical diagnoses. We report a new electrochemical immunosensor for ultrasensitive detection of phosphorylated p53 at Ser392 (phospho-p53(392)) based on graphene oxide (GO) as a nanocarrier in a multienzyme amplification strategy. Greatly enhanced sensitivity was achieved by using the bioconjugates featuring horseradish peroxidase (HRP) and p53(392) signal antibody (p53(392)Ab(2)) linked to functionalized GO (HRP-p53(392)Ab(2)-GO) at a high ratio of HRP/p53(392)Ab(2). After a sandwich immunoreaction, the HRP-p53(392)Ab(2)-GO captured onto the electrode surface produced an amplified electrocatalytic response by the reduction of enzymatically oxidized thionine in the presence of hydrogen peroxide. The increase of response current was proportional to the phospho-p53(392) concentration in the range of 0.02-2 nM with the detection limit of 0.01 nM, which was 10-fold lower than that of the traditional sandwich electrochemical measurement for p53(392). The amplified immunoassay developed in this work shows acceptable stability and reproducibility, and the assay results for phospho-p53(392) spiked in human plasma also show good recovery (92-103.8%). This simple and low-cost immunosensor shows great promise for detection of other phosphorylated proteins and clinical applications.
- Fullerenes Nanotubes and Carbon Nanostructures 01/2014; 23(5):410-417. · 0.64 Impact Factor
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ABSTRACT: Here a new electrochemical immunoassay platform has been developed for the trace amounts of myelocytomatosis (c-Myc) oncoprotein detection to solve the urgent need for improved early diagnostic tools of cancer. To our knowledge, this is the first electrochemical immunosensor for c-Myc oncoprotein determination by employing gold nanoparticles as labels in a signal enhancement strategy. It is based on a sandwich immunoassay where the target c-Myc oncoprotein (CAg) is captured by the primary c-Myc antibody (CAb) modified on gold substrate, followed by adding another CAb conjugated to gold nanoparticle tags. The proposed sensor shows a linear range between 4.3 pmol L−1 and 43 nmol L−1, with an estimated detection limit of 1.5 pmol L−1. The recovery was achieved between 96.2% and 109% in 1% serum samples. This sensitive immunosensor holds great promise for the early diagnostic application of cancer at a curable stage.Sensors and Actuators B Chemical 05/2013; 181:835-841. · 3.84 Impact Factor
- Sensors and Actuators B Chemical 02/2014; 191:396-400. · 3.84 Impact Factor
Published:January 6, 2011
r2011 American Chemical Society
dx.doi.org/10.1021/ac101715s|Anal. Chem. 2011, 83, 746–752
Functionalized Graphene Oxide as a Nanocarrier in a Multienzyme
Labeling Amplification Strategy for Ultrasensitive Electrochemical
Immunoassay of Phosphorylated p53 (S392)
Dan Du,†,‡Limin Wang,‡Yuyan Shao,‡Jun Wang,‡Mark H. Engelhard,‡and Yuehe Lin*,‡
†Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University,
Wuhan 430079, People's Republic of China
‡Pacific Northwest National Laboratory, Richland, Washington 99352, United States
ABSTRACT: P53 phosphorylation plays an important role in
many biological processes and might be used as a potential
biomarker in clinical diagnoses. We report a new electrochemi-
cal immunosensor for ultrasensitive detection of phosphory-
lated p53 at Ser392 (phospho-p53392) based on graphene oxide
(GO) as a nanocarrier in a multienzyme amplification strategy.
Greatly enhanced sensitivity was achieved by using the biocon-
jugates featuring horseradish peroxidase (HRP) and p53392
signal antibody (p53392Ab2) linked to functionalized GO (HRP-p53392Ab2-GO) at a high ratio of HRP/p53392Ab2. After a
sandwich immunoreaction, the HRP-p53392Ab2-GO captured onto the electrode surface produced an amplified electrocatalytic
fold lowerthanthat ofthe traditionalsandwichelectrochemicalmeasurement for p53392. Theamplifiedimmunoassay developedin
good recovery (92-103.8%). This simple and low-cost immunosensor shows great promise for detection of other phosphorylated
proteins and clinical applications.
cell growth and modulating DNA repair processes.1-3Loss of p53
It has been reported that p53 can be stabilized and accumulated in
the tumor cell nucleus as the result of p53 phosphorylation. There
are a few data concerning the measurement of p53 protein phos-
implication of p53 phosphorylation in human cancers.10,11For
example, Bar's group evaluated expression of p53 protein phos-
lated at Ser392 could be considered an independent prognostic
factor in advanced human esophageal squamous cell carcinomas
(ESCCs).11A recent study reported that p53 phosphorylated on
serine 15 might be used as a potential biomarker of γ-radiation
exposure since the phosphorylation is dose-dependent.13On the
basis of these results, quantitative determination of phosphorylated
p53 protein is enzyme-linked immunosorbant assay (ELISA).13,14
Although ELISA is a powerful tool for the detection of antigens and
he p53 protein, a well-known tumor suppressor and a
transcription factor, plays an important role in controlling
limits, it involves several incubation and washing steps followed
by spectrophotometric detection using a chromogenic substrate. In
addition, ELISA needs more expensive instruments and is lab-
oriented. In comparison with the ELISA method, electrochemical
immunoassay has attracted considerable interest because of its
intrinsic advantages such as portability, low cost, high sensitivity,
tumor markers.15-17In order to meet the increasing demand for
fication technologies using nanomaterials have been developed: (1)
Metal and semiconductor nanoparticles are directly used as electro-
particles are used as carriers to load a large amount of electroactive
species, such as ferrocene, to amplify the detection signal.20-23(3)
One of the most popular strategies is enzyme-functionalized nano-
a large amount of enzyme toward an individual sandwich immuno-
logical reaction event. Various nanomaterials have been used as
carriers to load enzymes and antibodies including carbon nano-
June 29, 2010
December 9, 2010
dx.doi.org/10.1021/ac101715s |Anal. Chem. 2011, 83, 746–752
nanoparticles,28and carboxylated magnetic beads.29For example,
Rusling's group24,25has achieved greatly enhanced sensitivity using
bioconjugates featuring horseradish peroxidase (HRP) labels and
signal antibodies linked to CNTs for immunodetection of the
prostate-specific antigen and interleukin-6, respectively. Lai et al.27
designed a novel tracer by one-pot assembly of glucose oxidase and
antibodies on gold nanoparticles for ultrasensitive multiplexed
immunosensor for R-fetoprotein detection based on carbon nano-
excitement in recent years and potential applications in sensors,
a few research groups have explored biological applications of
graphene and graphene oxide (GO) for cellular imaging and drug
delivery.38-41They found that the loading ratio (the weight ratio of
loadeddrug tocarriers) of GOcould reach200%, much higher than
that of other nanocarriers such as nanoparticles that usually have
a loading ratio less than 100%. Their work demonstrates that
advantages of large surface area, good biocompatibility, and physio-
logical stability. However, the controlled loading of enzymes and
antibodies to be designed as a tracer in immunosensing detection
remains relatively unexplored. In comparison to other carbon
easier to facilitate biomolecules binding via EDC chemistry.40,41
Our present work is motivated by the promising applications
of enzyme-functionalized nanoparticles in signal amplification
for ultrasensitive detection of biomarkers. Herein, the GO is
prepared by oxidizing graphite according to the modified Hum-
mer's method42-44and employed as a nanocarrier for enzyme
and antibody coimmobilization. Greatly amplified sensitivity is
achieved by using the bioconjugates featuring HRP and phospho-
p53392signal antibody (p53392Ab2) linked to functionalized GO via
amidization to the carboxylate groups of GO at a high HRP/
p53392Ab2ratio. For immunosensor fabrication, a gold nanoparticles
(AuNPs) modified screen-printed carbon electrode (SPCE) is used
as sensor platform to self-assemble a layer of N-hydroxysuccinimide-
activated hexa(ethylene glycol) undecane thiol (NHS) for primary
eactions, the HRP-p53392Ab2-GO conjugate is captured onto the
performed in the presence of hydrogen peroxide (H2O2) and
thionine (Scheme 1). Results demonstrated that the immunosensor
based on this amplification strategy has good dynamic range from
0.02 to 2 nM and low detection limit (0.01 nM) for phosphorylated
p53-S392 (phospho-p53392). It shows great promise for application
in biomedical research, clinical diagnosis, and screening radiation
Reagents and Materials. The human phospho-p53 (S392)
p53392antigen, biotin-phospho-p53392detection antibody, and
streptavidin-HRP, the human phospho-p53 (S15) ELISA kit,
and the human phospho-p53 (S46) ELISA kit were purchased
from R&D Systems Inc. Graphite powder (<45 mm), bovine
serum albumin (BSA), Triton X-100, Tween-20, 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hy-
droxysuccinimide (NHS), sodium chloroacetate (ClCH2COONa),
thionine, 3,30,5,50-tetramethylbenzidine (TMB), phosphate buffer
(ethylene glycol) undecane thiol (NHS) was obtained from Nano-
Science Instrument Inc.
Apparatus. Electrochemical experiments, including cyclic
with an electrochemical analyzer CHI 660A (CH Instruments,
Austin, TX) connected to a personal computer. Disposable gold
nanoparticles modified screen-printed carbon electrodes (AuNPs-
SPCE) were purchased from Dropsens Inc. A sensor connector
(Dropsens Inc.) was used to connect the disposable AuNPs-SPCE
with the CHI electrochemical analyzer. Confocal imaging was
performed at a Zeiss LSM 710 NLO laser scanning confocal
microscope with an upright Zeiss Axioexaminer stand. Transmission
TEM grids on a JEOL JSM-2010 TEM microscope operated at 200
kV. X-ray photoelectron spectroscopy (XPS) measurements were
taken with a Physical Electronics Quantum 2000 scanning micro-
Xpert X-ray diffractometer with Cu KR radiation at λ = 1.54 Å.
UV-vis measurements were carried out at room temperature on a
Safire 2 microplate reader (Tecan, Switzerland).
Synthesis of Graphene Oxide. GO was prepared according
of fuming HNO3was slowly added to the mixture under ice-cooling
and stirring. An amount of 25 g of KMnO4was slowly added under
ice-cooling and stirring. The mixed slurry was stirred at room
temperature for 120 h. After that, 600 mL of deionized (DI) water
was slowly added into the reacted slurry and stirred for 2 h; then 30
bright yellow solution with bubbling. The resultant solution was
and then washed in 1000 mL of DI water with 5 mL of HCl (37%)
centrifuged and then washed again. This process was repeated three
DI water until the pH of the washing solution increased to neutral
(∼6.5) (it took about 500 mL ? 12 washes). The remaining dark-
a fine powder. The dry process for GO must be carried out at low
temperatures because it slowly decomposes (deoxygenates) above
Scheme 1. Schematic Illustration of the Multienzyme Label-
ing Amplification Strategy Using HRP-p53392Ab2-GO
dx.doi.org/10.1021/ac101715s |Anal. Chem. 2011, 83, 746–752
60-80 ?C.45Then 1.0 mg mL-1GO aqueous solution was placed
into an ice bath to carry out strong sonication. The ice bath was
changed after each treatment to make sure that sample temperature
was below 5 ?C. At last, the resultant sample was centrifuged at 12 k
rgb for 10 min, and the upper solution was taken for future
Preparation of HRP-p53392Ab2-GO Conjugate. To convert
ester, hydroxyl, and epoxide groups to carboxylic groups, 50 mg of
mL-1GO suspension, followed by bath sonication for 2 h. After
these treatments, the resulting product GO-COOH was neutra-
lized with dilute hydrochloric acid and purified by repeated rinsing
water. Then, the GO-COOH suspension was dialyzed against
distilled water for over 48 h to remove any ions. For synthesis of
HRP-p53392Ab2-GO conjugate, HRP and p53392Ab2were at-
tached to carboxylated GO using anEDC/NHS amidizationproto-
col with a reaction mixture of 200/1 HRP/p53392Ab2molar ratio.
Briefly, the GO-COOH was mixed with 400 mM EDC and 100
mM NHS in 1 mL of pH 5.2 MES buffer and activated for 30 min.
The mixture was centrifuged at 13000 rpm for 5 min, and the
excess EDC and NHS. Subsequently, the resulting functionalized
mixture was dispersed in 1.0 mL of pH 7.4 PBS and sonicated for
5 minto obtaina homogeneous dispersion. Then, 50μL of HRP at
dispersion, and the mixture was stirred overnight at 4 ?C. The
reaction mixture was washed with PBS and centrifuged at 13000
rpm for 5 min three times, and the supernatant was discarded. The
resulting mixture was redispersed in 1.0 mL of pH 7.4 PBS contain-
gate is stable and can keep enzyme activity for at least 3 weeks.
Fabrication of the Immunosensor. An amount of 5 μL of
2 mg mL-1N-hydroxysuccinimide-activated hexa(ethylene glycol)
undecane thiol (NHS) was placed on the AuNPs-SPCE at room
temperature for 2 h to form a self-assembled layer. After thoroughly
washing with water, the electrode was immediately followed by
incubation with 5 μL of 90 μg mL-1phospho-p53392capture
antibody (Ab1) in pH 7.4 PBS for 1 h. After washing with 0.05%
Tween-20 and PBS buffer, the Ab1/NHS/AuNPs-SPCE was
incubated in 3% BSA and PBS solution at 37 ?C for 1 h to block
electrode was then washed with 0.05% Tween-20 and PBS buffer
Immunoassay Procedure for Detection of Phospho-p53392.
A sandwich immunoassay was used for determination of phospho-
p53392. (1) The immunosensor, Ab1/NHS/AuNPs-SPCE, was
incubated with 5 μL of a different concentration of phospho-p53392
20 and PBS buffer. (2) Next, the electrode (phospho-p53392/Ab1/
NHS/AuNPs-SPCE) was incubated with 5 μL of HRP-p53392-
Ab2-GO dispersion for 60 min, followed by washing with 0.05%
Tween-20 and PBS buffer to remove the nonspecific adsorption of
buffer solution containing 25 μM thionine and 2 mM H2O2.
’RESULTS AND DISCUSSION
Preparation and Functionalization of GO Conjugate. We
developed a simple and convenient route to prepare HRP-
p53392Ab2-GO conjugates, as shown in Scheme 2. Briefly, water-
soluble GO was first synthesized by oxidizing graphite according to
the modified Hummer's method,42-44followed by strong sonica-
tion to disperse GO and by centrifugation to remove nondispersed
large GO layers. The TEM image provides more detailed morpho-
typical wrinkle morphology of GO and is exfoliated into single or
very thin layers. Sonication treatment of the GO truncated it from
the microscopy image of GO, and Figure 1C shows its size distri-
GO (curve b). It can be seen that, after oxidation, the sharp diffrac-
tion peak in graphite (2θ = 26.5?, corresponding to the interlayer
Scheme 2. Schematic Illustration of Preparation of
Figure 1. (A) TEM image of the GO; (B) microscope image of
synthesized GO; (C) size distribution of GO.
dx.doi.org/10.1021/ac101715s |Anal. Chem. 2011, 83, 746–752
distance d = 0.336 nm) disappeared, and a new diffraction peak
(2θ = 11.4?, d = 0.777 nm) appeared in GO,46indicating the
exfoliating GO and obtaining single-layer or few-layer graphene
sheets.47Graphite and GO are further characterized with XPS, and
the results are shown in Figure 2B. It can be seen that graphite
exhibits a single sharp C1s XPS spectrum centered at 284.3 eV
(mainly sp2carbon, curve a). GO shows a typical C1s spectrum of
heavily oxygenated carbon (curve b). In our previous study, we
GO than that in CNT and graphite.44
There are ester, hydroxyl, and epoxide groups on the GO
surface.38,39In order to facilitate the binding of enzymes and anti-
bodies to the GO via the EDC/NHS method, it is necessary to
convert these groups into carboxyl groups for biomolecule loading.
In our strategy, the GO was mixed with ClCH2COONa under
strong basic conditions. According to literatures,38,41the ester,
hydroxyl, and epoxide groups on the GO were converted into
tion mixture, the precipitated GO-COOH was well-dispersed in
was achieved through formation of an amide bond by the reaction
between the NH2groups of the biomolecules and the COOH of
at 399.6 eV was observed on the HRP-p53392Ab2-GO conjugate
(curve b). This typical binding energy of amide nitrogen atoms
(HN-CdO) indicated successful functionalization of GO to form
Ab2-GO was further characterized by UV-vis spectroscopy, as
shown in Figure 3B. For pure p53392Ab2and HRP, they displayed
absorption peaks at 282 nm (curve a) and 404 nm (curve b),
When p53392Ab2and HRP were bound to GO via the EDC/NHS
method, two obvious absorption peaks were observed on the
resulting HRP-p53392Ab2-GO conjugate (curve d), indicating
successful binding of p53392Ab2and HRP to GO.
Signal Amplification Immunoassay with HRP-p53392Ab2-
GO Conjugate. To enhance detection sensitivity, herein we pur-
during the immunoassay. For the immunosensor fabrication, phos-
modified SPCE (AuNPs-SPCE) through a layer of N-hydroxysuc-
cinimide-activated hexa(ethylene glycol) undecane thiol (NHS). As
shown in Figure 4A, the cyclic voltammograms at AuNPs-SPCE
AuNPs-SPCE exhibited a pair of stable and well-defined redox
peaks at -0.225 and -0.247 V (curve c), respectively, which corre-
spond to the electrochemical oxidation and reduction of thionine.
When the immunosensor was incubated with 1 ng mL-1phospho-
p53392antigen, no obvious change in response was observed at the
sensor (data not shown). However, after incubating with biotin-
p53392Ab2for 1 h and then streptavidin-HRP for 20 min, the
Ab1/NHS/AuNPs-SPCE displayed an obvious increase in electro-
catalytic reduction current (curve d) because of the introduction of
HRP onto the electrode surface by the immunoreactions. Further-
more, we observed a more significant increased reduction current at
the immunosensor (curve e) when replacing HRP-streptavidin-
biotin-p53392Ab2with the HRP-p53392Ab2-GO conjugate dur-
ing the sandwich immunoreactions. It is not surprising that the
multienzyme labeling strategy enhanced detection responses com-
pared with single-enzyme-labeled antibody in the conventional
immunoassay. The achieved amplification of signal was ascribed to
a large amount of enzymes loaded on the GO nanocarrier.
Because of the coimmobilization of enzymes and antibodies
on the GO nanocarrier, the ratio of HRP and p53392Ab2(HRP/
p53392Ab2) is the most important factor on the response signal. As
shown in Figure 4B, one can see that the electrocatalytic current
mum response is achieved at the ratio of 200/1. The increase of the
Figure 2. (A) XRD patterns of (a) graphite and (b) GO; (B) XPS
measurements of C1s from (a) graphite and (b) GO.
Figure 3. (A) XPS measurements of N1s from (a) HRP-p53392Ab2-
GO and (b) GO. (B) UV-vis spectra of (a) p53392Ab2, (b) HRP, (c)
GO, and (d) HRP-p53392Ab2-GO conjugate.
Figure 4. (A) Cyclic voltammograms obtained at (a) AuNPs-SPCE,
(b) Ab1/NHS/AuNPs-SPCE in pH 7.4 PBS, (c) Ab1/NHS/AuNPs-
SPCE, (d) HRP-streptavidin-biotin-p53392Ab2/phospho-p53392/
Ab1/NHS/AuNPs-SPCE, (e) HRP-p53392Ab2-GO/phospho-p53392/
mM H2O2. (B) Effect of HRP/p53392Ab2ratio on response current. A
concentration of 1 ng mL-1phospho-p53392antigen was used during the
dx.doi.org/10.1021/ac101715s |Anal. Chem. 2011, 83, 746–752
HRP/p53392Ab2ratio could increase the total amount of HRP
loaded per GO nanocarrier, which is expected to enhance the
response amplification for this sandwich immunoassay. However,
at the electrode surface, which may result in a decreased response.
Therefore, the HRP/p53392Ab2ratio of 200/1 is selected as the
To determine the amount of active HRP in the HRP-
p53392Ab2-GO conjugate dispersion, the mixture was reacted
with HRP substrate TMB. The reaction product was read at 650
nm. These results were compared to a standard curve con-
structed with pure HRP by an enzyme activity experiment. The
concentration of active HRP in the HRP-p53392Ab2-GO
conjugate dispersion was determined to be 4.77 μg mL-1.
Optimization of Detection Conditions. Nonspecific adsorp-
tion has a significant influence on immunoassay responses. A series
of corresponding control experiments (Ab1/NHS/AuNPs-SPCE
was directly exposed to HRP-p53392Ab2-GO conjugate in the
absence of phospho-p53392antigen) using different concentrations
of BSA blocking were performed in PBS containing thionine and
H2O2by SWV measurements. As shown in Figure 5A, the current
responses at both the HRP-p53392Ab2-GO/phospho-p53392/
Ab1/NHS/AuNPs-SPCE and corresponding control HRP-
p53392Ab2-GO/Ab1/NHS/AuNPs-SPCE decreased upon in-
effect of BSA. The minimization of the nonspecific adsorption
tended to be stable after that; however, the immunsensing signals
to 5%. The decrease of signal may be due to the steric hindrance of
large molecules, which block or hinder the diffusion of substrate
toward the electrode surface and the electron-transfer reaction.
Although the immunosensor displayed much higher electrochemi-
signal was very high. Considering the sensitivity of the electro-
the nonspecific adsorption, 3% BSA in PBS was selected as the
The incubation time is another important parameter for both
capturing phopho-p53392antigen and specifically recognizing
HRP-p53392Ab2-GO. It can be seen that the electrochemical
response increases with increasing incubation time of phospho-
p53392antigen and tends to a steady value after 1 h (curve a in
Figure 5B), indicating a thorough capturing of the antigens on
the electrode surface. In the second immunoassay incubation
step, the catalytic current also increases upon increasing the
incubation time and reaches a plateau at 1 h, which shows
saturated binding sites between antigen and detection antibody
(curve binFigure5B). Alongertimeincubationcould resultina
large nonspecific signal. Therefore, the optimal incubation time
for the first and second immunoreactions is 1 h, respectively.
Analytical Performance of the Immunosensor for Electro-
chemical Detection of Phospho-p53392Antigen. After the
above optimizations, the proposed immunosensor using HRP-
p53392Ab2-GO conjugate in the amplification approach is chal-
Figure 6A. It can be seen that the SWV currents increase with the
increase of phospho-p53392concentrations. The linear response is
obtained over the concentration range from 0.02 to 2 nM with the
detection limit of 0.01 nM (curve a in Figure 6B). Comparably, we
used the traditional HRP-streptavidin-biotin-p53392Ab2 for
phospho-p53392determination on electrode. The increase of re-
duction current is also proportional to the phospho-p53392con-
centration in the range of 0.1-2 nM with the detection limit of 0.1
labeling amplification strategy using GO as a nanocarrier possesses
that of the traditional labeling immunoassay. The detection limit of
this immunosensor is 10-fold lower than that of a conventional
sensor with the HRP-streptavidin-biotin-p53392Ab2label. The
achieved high sensitivity mainly results from the excessive enzymes
present in the GO. The phospho-p53 (S392) ELISA kit shows a
linear range from 0.05 to 3 nM with detection limit of 0.05 nM for
phospho-p53392. This amplified electrochemical immunosensor is
chemical detector used in this work is much simpler and lower in
cost compared with the instrument used in the ELISA kit.
To further investigate the selectivity and validate the sensor
performance for phospho-p53392detection, the proposed im-
munosensor was tested using human plasma as matrix. The
immunosensor was incubated in human plasma samples spiked
with 1.0 ng mL-1phospho-p53392and different possible inter-
fering agents such as p53, phospho-p5315, and phospho-p5346.
No remarkable electrochemical response change was observed
the presence of phospho-p53392, indicating good selectivity for
determination of phospho-p53392.
of (red bars) HRP-p53392Ab2-GO/phospho-p53392/Ab1/NHS/
AuNPs-SPCE and (green bars) HRP-p53392Ab2-GO/Ab1/NHS/
AuNPs-SPCE control. (B) Incubation time for (a) capturing phopho-
p53392antigen and (b) recognizing HRP-p53392Ab2-GO on the
Figure 6. (A) SWV curves acquired at HRP-p53392Ab2-GO/phospho-
p53392/Ab1/NHS/AuNPs-SPCE after incubation with (a) 0, (b) 0.01,
(c) 0.02, (d) 0.05, (e) 0.1, (f) 0.2, (g) 0.5, (h) 1, (i) 2, and (j) 5 ng mL-1
phospho-p53392antigen in pH 7.4 PBS containing 25 μM thionine and
AuNPs-SPCE for detecting phospho-p53392antigen.
dx.doi.org/10.1021/ac101715s |Anal. Chem. 2011, 83, 746–752
The reproducibility of the proposed immunosensor is evalu-
ated by intra- and interassay coefficients of variation (CVs). The
intra-assay precision of the analytical method is evaluated by
analyzing one immunosensor for six replicate determinations.
The CVs of the intra-assay were 3.3% and 4.6% at 0.1 and 1.0 ng
mL-1phospho-p53392, respectively. Similarly, the interassay
CVs on six immunosensors were 3.9% and 5.7% at 0.1 and 1.0
ng mL-1phospho-p53392, respectively. These results demon-
strated acceptable reproducibility and precision of the proposed
immunosensor. In addition, the immunosensor could be stored
at 4 ?C. In this way, over 90% of the initial response remained
after 1 week and 80% of the initial response remained after 1
month, indicating acceptable stability.
A series of phospho-p53392human plasma samples were used
to test the accuracy of the electrochemical quantification ap-
proach. Phospho-p53392human plasma samples were prepared
by spiking different amounts of phospho-p53392with known
concentrations to human plasma. The results are summarized in
Table 1, which shows the recoveries are in the range of
92-104%, indicating that the electrochemical immunosensing
approach is reliable.
In summary, we have successfully designed a multienzyme
labeling GO strategy in a signal amplification procedure and
demonstrated its use in the ultrasensitive, selective, and accurate
quantification of phospho-p53392by electrochemical immunoas-
say. Enhanced sensitivity is achieved by using functionalized GO
as a nanocarrier to link enzyme and signal antibody at high ratio.
The proposed immunosensor shows excellent performance for
detectionofphosphorylatedprotein withawidelinearrange and
low detection limit and acceptable stability, reproducibility, and
accuracy. We anticipate that this method can be extended for
determination of other proteins and provide a promising poten-
tial in clinical applications.
The work was done at Pacific Northwest National Laboratory
Health CounterACT program through the National Institute of
PNNL Laboratory Directed Research and Development program.
Its contents are solely the responsibility of the authors and do not
necessarily represent the official views of the federal government.
PNNL is operated for the U.S. Department of Energy (DOE) by
Battelle under contract DE-AC05-76RL01830. The materials char-
Laboratory, a national scientific user facility sponsored by DOE's
office of Biological and Environmental Research located at PNNL.
Dan Du acknowledges the support from the National Natural
Science Foundation of China (21075047) and the Program for
Chenguang Young Scientist for Wuhan (200950431184).
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