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j.bios.2017.10.048

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Author’s Accepted Manuscript
Highly sensitive electrochemical nuclear factor
kappa B aptasensor based on target-induced dualsignal ratiometric and polymerase-assisted protein
recycling amplification strategy
Kanfu Peng, Pan Xie, Zhe-Han Yang, Ruo Yuan,
Keqin Zhang
www.elsevier.com/locate/bios
PII:
DOI:
Reference:
S0956-5663(17)30715-7
https://doi.org/10.1016/j.bios.2017.10.048
BIOS10073
To appear in: Biosensors and Bioelectronic
Received date: 3 September 2017
Revised date: 23 October 2017
Accepted date: 26 October 2017
Cite this article as: Kanfu Peng, Pan Xie, Zhe-Han Yang, Ruo Yuan and Keqin
Zhang, Highly sensitive electrochemical nuclear factor kappa B aptasensor based
on target-induced dual-signal ratiometric and polymerase-assisted protein
recycling
amplification
strategy, Biosensors and Bioelectronic,
https://doi.org/10.1016/j.bios.2017.10.048
This is a PDF file of an unedited manuscript that has been accepted for
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Highly sensitive electrochemical nuclear factor kappa B aptasensor
based on target-induced dual-signal ratiometric and
polymerase-assisted protein recycling amplification strategy
Kanfu Penga, Pan Xiea, Zhe-Han Yangb, Ruo Yuanb, Keqin Zhanga, 
a
Department of Kidney, Southwest Hospital, The Third Military Medical University,
Chongqing 400038, China
b
Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education,
College of Chemistry and Chemical Engineering, Southwest University, Chongqing
400715, China
ABSTRACT: In this work, an amplified electrochemical ratiometric aptasensor for
nuclear factor kappa B (NF-κB) assay based on target binding-triggered ratiometric
signal readout and polymerase-assisted protein recycling amplification strategy is
described. To demonstrate the effect of “signal-off” and “signal-on” change for the
dual-signal electrochemical ratiometric readout, the thiol-hairpin DNA (SH-HD)
hybridizes with methylene blue (MB)-modified protection DNA (MB-PD) to form
capture probes, which is rationally introduced for the construction of the assay
platform. On the interface, the probes can specifically bind to target NF-κB and
expose a toehold region which subsequently hybridizes with the ferrocene
(Fc)-modified DNA strand to take the Fc group to the electrode surface, accompanied

Corresponding authors at: Tel./ fax: +86 23-68754188; E-mail addresses: zhkq2000@sina.com (K.Q. Zhang).
1
by displacing MB-PD to release the MB group from the electrode surface, leading to
the both “signal-on” of Fc (IFc) and “signal-off” of MB (IMB). In order to improve the
sensitivity of the electrochemical aptasensor, phi29-assisted target protein recycling
amplification strategy was designed to achieve an amplified ratiometric signal. With
the above advantages, the prepared aptasensor exhibits a wide linear range of 0.1 pg
mL-1 to 15 ng mL-1 with a low detection limit of 0.03 pg mL-1. This strategy provides
a simple and ingenious approach to construct ratiometric electrochemical aptasensor
and shows promising potential applications in multiple disease marker detection by
changing the recognition probe.
Keywords: Electrochemical aptasensor; Dual-signal ratiometric; Nuclear factor kappa
B assay;Polymerase-assisted protein recycling amplification

1. Introduction
The sensitive detection of disease-related protein plays an important role in clinical
diagnostics and treatment (Li et al., 2016; Bell et al., 2015). Until now, various
techniques such as colorimetry (Gupta et al., 2016; Wu et al., 2016), electrochemistry
(Dutta et al., 2017; Dutta et al., 2015), spectroscopy (Casalini et al., 2015; Adányi et
al., 2017) and molecular biology have been implemented toward protein detection.
Alternatively, electrochemical biosensors are particularly attractive because of their
remarkable advantages including high sensitivity, simplicity, good selectivity and low
cost (Lin et al., 2015; Kokkinos et al., 2016). The conventional electrochemical
2
biosensor have been proposed by designing artful target protein-induced
electrochemical “signal-on” or “signal-off” changes on the electrode surface (Li et al.,
2016; Wang et al., 2015). However, only depend on a single “signal-on” or “signal-off”
changes, it is difficult to confirm whether the observed the signal change is due to
target binding or other physical and chemical reactions, which can lead to relatively
low reproducibility, the false-positive and the false-negative test result.
Recently, the ratiometric techniques that utilize two labels simultaneously for
robust detection of one specific target have been identified to be a good method to
avoid other physical and chemical influence for biosensor. Among various ratiometric
methods (e.g. fluorescence, electrochemistry and chemiluminescence) (Špringer et
al., 2013; Yan et al., 2015; Shen, et al., 2015), the ratiometric electrochemical
biosensor has been aroused considerable interest owing to its remarkable advantages
including high sensitivity, simplicity and relatively low cost (Cai et al., 2016; Ren et
al., 2013). Over the past decades, three types of ratiometric electrochemical
biosensors have been reported. The first type is two-channel ratiometric detection that
was conducted by implementing two working electrodes (Zhang et al., 2015; Chai et
al., 2013). However, this method are not only complicated but also difficult to ensure
the close surface/interface conditions of the signal probe (sP) and inner reference
probe (rP) between two electrodes, leading to significant detection error. To overcome
these shortcomings, the second type of electrochemical ratiometric biosensor was
constructed by simultaneous immobilization of sP and rP at one sensing interface (Wu
et al., 2013; Deng et al., 2017). Although this ratiometric sensor is relatively simple
3
and accurate in comparison with the two-channel ratiometric detection, the observed
signal changes that are due to target binding or deterioration of the sensing surface is
doubtful because rP cannot provide the in situ information on sP owing to the
independency between sP and rP, which limits the practical applications of the
ratiometric sensor. In the view of this defect, the third type of electrochemical
ratiometric biosensor was developed by introducing two different sP to obtain
“signal-on” and “signal-off” change simultaneously. For example, Ju group designed
an immunoreaction-triggered ratiometric sensor by using proximity hybridization to
form a sequence for opening ferrocene (Fc)-labeled hairpin DNA that was
immobilized on electrode surface and introducing methylene blue (MB)-labeled
DNA-antibody (Ren et al., 2015). However, the reported approaches to trigger
“signal-off” and “signal-on” change for the dual-signal ratiometric are complex and
the reported biosensor suffer from low sensitivity because a single target only opens a
single signaling capture probe and makes a pair of Fc/MB labeled probe
conformational change. Therefore, exploring an efficient ratiometric electrochemical
biosensor that can solve the existing problems and achieve higher accuracy for
ratiometric detection is urgently needed.
In this work, an electrochemical ratiometric aptasensor based on target
binding-triggered ratiometric signal readout and polymerase-assisted protein recycling
amplification strategy was developed for nuclear factor kappa B (NF-κB)
determination. The detailed principle is illustrated in Scheme 1. Three DNA strands
including the protection DNA (PD) labeled with MB on the 5’ and blocked with
4
(CH2)6 spacers (MB-PD-(CH2)6) on the 3’, the trigger DNA (TD) modified with Fc
(Fc-TD) on the 5’ and the thiol-modified hairpin DNA (SH-HD) with the sequence of
NF-KB aptamer (the red part in Scheme 1) are programmed to fabricate the biosensor.
The target binding-induced ratiometric signal readout was performed as follows.
Firstly, the SH-HD/MB-PD probes are formed by hybridizing SH-HD with MB-PD,
and then self-assembled on electrode surface. In the presence of target NF-κB,
sequence of NF-κB aptamer in probes specifically bind to the NF-κB and expose a
short sequence which is used as a toehold to hybridize with Fc-TD. Then, strand
displacement reaction is driven between Fc-TD and MB-PD in SH-HD/MB-PD with
formation of SH-HD/Fc-TD duplex. Thus, the MB is released from the electrode
surface and the Fc is introduced into the electrode surface which leads to the decrease
of the current response of MB (IMB), accompanied by the increment of that of the Fc
(IFc). As a result, “signal-off” and “signal-on” elements for dual-signal
electrochemical
ratiometric
readout
are
achieved.
To
enhance
sensitivity,
polymerase-assisted protein recycling amplification strategy is designed with aid of
phi29. The Phi29 bind to 3’-OH of Fc-TD in the formed SH-HD/Fc-TD duplex to
extend from 3’ to 5’of Fc-TD, leading to the target NF-κB to release from
SH-HD/Fc-TD and take part in the next cycle. As a result, the much more MB tags
were away from the electrode surface and the much more Fc tags were introduced into
the electrode surface, amplifying ratiometric signal. Based on the value of IFc/IMB,
NF-κB was detected sensitively. This ratiometric method holds a great potential for
the development of sensitive biosensing platform for protein assays.
5

2. Experiments Section
2.1 Materials
The target NF-κB standard solution was purchased from Elabscience Bio. Co. Ltd.
(Wuhan, China). The Hexanethiol (HT), gold chloride (HAuCl4·4H2O) and
Tris(2-carboxyethy)phosphine hydrochloride (TCEP) were obtained from Sigma (St.
Louis, MO, USA). Phi29 DNA polymerases (containing reaction buffer for phi29
catalyzed polymerization, donated as phi29 buffer) and deoxynucleotide solution
mixture (dNTPs) were supplied by New England Biolabs Co., Ltd. (Ipswich, USA).
Three HPLC-purified oligonucleotide sequences were purchased from Sangon
Biotech Co., Ltd (Shanghai, China), and the sequences are listed below: the hairpin
DNA (HD), 5′-AC CTG GGA AAG TCC CCA GGT GA CTG CAA TCG TTC
A-(CH2)6-SH-3′; protection DNA (PD): 5′-MB-TGA ACG ATT GCA GTC-(CH2)6-3′;
Trigger DNA (TD): 5′-Fc-TGA ACG ATT GCA GTC ACC TGG-3′.
2.2. Apparatus and Measurements
A modified glassy carbon electrode (GCE), a platinum wire auxiliary electrode
and a saturated calomel reference electrode (SCE) were used as a three-compartment
electrochemical cell. Electrochemical measurement including cyclic voltammetry
(CV), differential pulse voltammetry (DPV) and electrochemical impedance spectra
(EIS) were conducted by a CHI 660D electrochemical workstation (Shanghai
Chenhua instrument, China). Gel electrophoresis was conducted using a DYY-8C
electrophoretic apparatus (Beijing, Wo De Life Sciences Instrument Co., Ltd, China).
6
2.3 Preparation of the electrochemical aptasensor
Firstly, GCE with a mirror-like surface was obtained by polishing it with 0.3 and
0.05 μm alumina powders and sonicating in ultrapure water and rinsed thoroughly.
After that, the clean GCE was soaked in HAuCl4 solution (1%, w/w) and
electrodeposited at -0.2 V for 30 s to obtain AuNP layer modified GCE (Au/GCE).
The mixture of thiolated hairpin DNA (HD, 1.5 μM) and protection DNA (PD, 2.0
μM) in 20 mM Tris-HCl buffer (100 mM NaCl, TCEP 1.0 mM, pH 7.4) was heated to
95 °C for 5 min and then allowed to cool down to room temperature for 60 min to
form the DNA probes. Then, the obtained DNA probes were immobilized on the
electrode surface by Au-S bond after incubation for 16 h at room temperature. Next,
HT solution (10 μL, 1%, w/w) was incubated on the probe modified electrode surface
for 40 min to obtain the HT/probe/Au/GCE. Subsequently, 20 μL of phi29 buffer (33
mM Tris-acetate, 10 mM Mg-acetate, 66 mM Kacetate,1 mM DTT, pH 7.5)
containing phi29 (100 U/mL), dNTPs (200 μM), TD (2.5 μM) and NF-κB standard
solution with different concentrations were dropped on the resulting electrode surface
and incubated at 37 °C for 2 h. To remove the physically adsorbed species, the
modified electrode was thoroughly cleaned with ultrapure water. DPV measurement
was used to carry out the detection experiment in PBS (pH 7.0), giving the
quantitative criteria for NF-κB assay.
2.4 Electrochemical Measurements
The DPV were conducted at a sweeping rate 50 mV s−1, 50 ms pulse width, 0.2 s
pulse period, and voltage range from −0.5 to 0.5 V. CV parameter was a scan rate of
7
100 mV/s. EIS was conducted over a frequency range from 10 kHz to 0.1 Hz using an
alternative voltage with an amplitude of 5 mV that superimposed on a dc potential of
0.22 V. The obtained EIS spectrums were fitted by using ZSimpWin electrochemical
impedance modeling software to a modified Randles equivalent circuit. Both the EIS
and CV measurements were recorded in a 0.01 M PBS buffer solution containing 5.0
mM [Fe(CN)6]3−/4− redox pair (1:1 molar ratio).
2.5 Non-denaturing polyacrylamide gel electrophoresis (PAGE)
The dynamic DNA assembly products were investigated by non-denaturing
polyacrylamide gel (16%), operated in 1× TBE buffer at 120 V for 120 min. Before
loading, DNA samples were mixed with DNA loading buffer on a volume ratio of 5:1.
The DNA samples in gel electrophoresis were stained in ethidium bromide (EB) dye
solution. After separation, the gels were scanned using the BIO-RAD gel image
analysis system (BIO-RAD, USA).

3.Results and discussions
3.1 Characterization of aptasensor
To characterize the aptasensor preparation procedure, electrochemical impedance
spectroscopy (EIS) measurement was conducted. As shown in Fig. 1A, the curve a is
the EIS response of bare GCE. Then, a decrease in the charge-transfer resistance (Rct)
value can be observed after electrodeposition of AuNPs on GCE (curve b) because the
conductivity of AuNPs accelerate the electron transfer of the redox probe to the
electrode. When the self-assembly of the probes are modified on Au/GCE surface by
Au-S bond, the Rct value increase owing to the negative charge of the probes (curve c).
8
A further increase of Rct can be observed when the surface is blocked with HT that
can obstruct the electron transfer of the redox probe to the electrode (curve d),
suggesting that the aptasensor is successfully prepared. Finally, the enzyme
assisted-target NF-κB recycling introduces larger amount of duplex DNA on modified
electrode surface, thus, an increase Ret value can be observed.
To further provide information about the preparation of the aptasensor, CV
measurements were performed to monitor the surface features of electrode in the
presence of 5.0 mM [Fe(CN)6]3−/4−. As shown in Fig. 2B, a pair of well-defined redox
peaks of [Fe(CN)6]3−/4− are obtained at bare GCE (curve a). When AuNPs are
electrodeposited on the electrode surface, the redox peak currents increase
significantly (curve b), which ascribes to the conductivity of AuNPs accelerate the
electron transfer. After the self-assembly of the probes are immobilized on Au/GCE,
an obvious decrease of peak current can be observed (curve c) due to the negative
charge of the probes. Subsequently, when the modified electrode is blocked with HT, a
further decrease peak current is obtained (curve d) because HT obstructs the electron
transfer of the redox probe to the electrode. Finally, the peak current decrease
apparently after the enzyme assisted-target NF-κB recycling (curve e). The reason for
that the generated duplex DNA obstruct electron transfer on the surface of electrode.
3.2 PAGE analysis
The enzyme assisted-target NF-κB recycling was verified by PAGE image. It can
be seen that the PD, TD and HD in lanes 1, 2, 3 respectively, exhibit a different single
band (Fig. 2). A band with obviously decreased migration shift is observed in lane 4
9
(lane 4 vs. 1, 3), which can be attributed to the hybridization products between HD
and PD, indicating that HD successfully hybridized to PD with formation of DNA
probe. When added TD, NF-Κb, Phi29 and dNTPs into the sample of DNA probe, a
band with very slow mobility is obtained (lane 5 vs. 4), which indicates that enzyme
assisted-target NF-κB recycling lead to the successful formation long dsDNA
molecule. The gel electrophoresis results confirms the feasibility of the target
binding-triggered strands replacement and then -induced polymerase-assisted protein
recycling amplification strategy.
3.3 Feasibility of the electrochemical aptasensor for NF-κB assay
To verify the feasibility of the proposed aptasensor for the assay of NF-κB, DPV
measurements were conducted on the HT/probe/Au/GCE in the presence/absence of
Phi29 and dNTPs with/without NF-κB. As shown in Fig.3, in the absence of both
NF-κB and Phi29, dNTPs (curve a), the oxidation peak current of MB can be
observed, in contrast, the oxidation peak current of Fc cannot be observed. In the
presence of only Phi29 and dNTPs, no obvious changes of the oxidation peak currents
of MB and Fc are observed (curve b). However, in the presence of NF-κB without the
addition of Phi29 and dNTPs, a decrease oxidation peak current of MB and an
increase oxidation peak current of Fc can be observed (curve c), indicating that the
DNA stands-replacements between MB-PD and Fc-TD are successfully triggered by
the binding of probes to NF-κB. When the HT/probe/Au/GCE is incubated with
NF-κB, Phi29 and dNTPs, significant changes of the peak currents of MB and Fc
(curve d) can be observed, owing to the high-efficient DNA stands-replacements
10
triggered by the recognition of NF-κB and the recycling of NF-κB.
3.4. Optimization of experimental conditions
To obtain optimal performance for amplified electrochemical determination of
NF-κB by the proposed method, the experimental parameters including the
immobilization concentration of the DNA probes and the phi29 assisted-target protein
recycling time were optimized. Due to the packing density of the DNA probes
anchored on gold electrode can intensely influence the analytical performance, the
effect of the immobilization concentration of the DNA probes was firstly investigated.
As shown in Fig. 4A, it can be seen that the value of IFc/IMB gradually increase with
increment immobilization concentration of DNA probes form 0.5 M to 1.5M.
However, when the concentration of the DNA probes further increase, a decrease
IFc/IMB value is observed. Such a decrease is possibly because high immobilization
concentration of the DNA probes induce steric hindrances and electrostatic repulsion,
which increases the unfavorable interaction between neighboring DNA probes such as
target protein recognition, DNA stand replacement and enzyme polymerization.
Therefore, the concentration of the DNA probes at 1.5 μM is selected for subsequent
experiments. The time of phi29 assisted-target protein recycling is another important
parameter influencing the performance of the designed aptasensor. As shown in Fig.
4B, the value of IFc/IMB increases with an extended the time of phi29 assisted-target
protein recycling from 0.5 to 2 h, and have no obvious change after the time reached
2.5 h, suggesting that 2 h is sufficient for the phi29 assisted-target protein recycling
during the NF-κB detection.
11
3.5 Electrochemical aptasensor performance analysis
The analytical performances of the proposed aptasensor were assessed. As shown in
Fig. 5A, it can be seen that the peak current of MB decrease and the peak current of
Fc increase with the increment of NF-κB concentration. The Fig. 5B shows the
dependence of the change of the oxidation peak current of MB (IMB) and Fc (IFc) on
the NF-κB concentration in the range of 0.1 pg mL-1 to 20 ng mL-1, respectively. Both
of them exhibit a linear 10 pg mL-1 to 15 ng mL-1 and the detection limits are 0.8 pg
mL-1 (based on Fc signal) and 0.6 pg mL-1 (based on MB signal) (S/N=3), respectively.
When using IFc/IMB as signal, the value of IFc/IMB is linear with the concentration of
NF-κB in the range from 0.1 pg mL-1 to 15 ng mL-1, with the linear regression
equation is log(IFc/IMB) =0.23 log cNF-κB + 0.0086 (R2 = 0.9865), and the detection
limit is 0.03 pg mL-1 (S/N = 3) (Fig. 5C). It can be found that the electrochemical
ratiometry exhibits a lower detection limit and a wider linear range than that obtained
by using IFc or IMB as detection signal alone. Moreover, the performance of proposed
aptasensor is better than that of the aptasensor based on radiometric method only
(Wang et al, 2017) or based on polymerase-assisted protein recycling amplification
strategy only (Wang et al, 2016). Furthermore, this method provides comparable
detection sensitivity compared with those reported electrochemical for NF-κB
detection (Table 1).
3.6 Selectivity, reproducibility and stability
After investigating the sensitivity of the proposed aptasensor for the NF-κB assay,
we then challenged its ability to selectivity, reproducibility and stability. To assess the
12
selectivity of the aptasensor, three possible interfering proteins including
immunoglobulin G (IgG), thrombin (TB) and procalcitonin (PCT) were investigated
under the same experimental conditions. As shown in Fig.S1, when the proposed
aptasensors are used to detect IgG (100 ng mL-1), TB (100 ng mL-1) and PCT (100 ng
mL-1) respectively, the DPV responses are almost no difference with that of blank test.
However, a high DPV response is achieved once the aptasensor incubated with 5 ng
mL-1 NF-κB. Meanwhile, when the aptasensor incubate with the mixture solution of 5
ng mL-1 NF-κB and the three interferences (100 ng mL-1), the DPV responses are
almost no obvious change in comparison with the case of only NF-κB. The above
experimental results indicate the high selectivity of this aptasensor for NF-κB
detection.
To investigate the reproducibility, five of the aptasensors with 5 ng mL-1 NF-κB
were assessed under the same detection condition (Fig.S2A). Similar DPV responses
are achieved and the relative standard deviation (RSD) is 9.1%. Besides, the same
aptasensor for detecting five times is also assessed and the RSD is 8.8% (Fig.S2B).
These two results demonstrates that the reproducibility is acceptable.
To evaluate the stability of the proposed aptasensor, the long-term stability
experiment was investigated by storing the aptasensor at 4 ºC and assaying every four
days (Fig.S2C). Five of aptasensors with 5 ng mL-1 NF-κB were evaluated under the
same detection conditions. After 20 days, the DPV responses retain 94.5%, 91.2%,
90.1%, 88.8% and 87.7% of their initial current, suggesting that the aptasensor has
good stability.
13
3.7 Clinical serum samples analysis
The analytical reliability and application potential of the newly developed
aptasensor for NF-κB assay was examined by recovery experiment. The aptasensor
incubated with the healthy human serum sample contained various concentrations of
NF-κB to assess the influence of the serum samples for detection NF-κB.
Corresponding to the results shown in Table 2, it can be see that the recovery is
varying from 90.0% to 103% and RSDs is ranging from 6.73% to 9.19%. Comparison
between the experimental results obtained by the proposed method and the
enzyme-linked immunosorbent assay (ELISA) was also performed via a least-squares
regression method. As shown in Table S1, good correlations were achieved after
comparison with the conventional assay for NF-κB in 5 human serum samples.
Assays on serum from normal individuals clearly suggested that using the developed
aptasensor to monitor NF-κB concetration in serum samples is promising.

4. Conclusion
In summary, we developed an amplified electrochemical ratiometric aptasensor for
NF-κB detection based on target binding-triggered ratiometric signal readout and
polymerase-assisted protein recycling amplification strategy. The binding between
target NF-κB and probes result in the effect of “signal-off” and “signal-on” change for
the dual-signal electrochemical ratiometric readout by strand displacement reaction.
Moreover, phi29-assisted target protein recycling amplification strategy is designed to
14
enhance the electrochemical ratiometric signal for improving the sensitivity of the
electrochemical aptasensor. Compared with the nonratiometric and conventional
ratiometric sensors, the proposed ratiometric electrochemical aptasensor exhibits
higher accuracy, robustness and sensitivity. The simplicity in operation together with
the excellent analytical performance of the aptasensor makes it useful and powerful
for applications in biochemical investigations. In view of these advantages, the
developed ratiometric electrochemical aptasensor may offer much better flexibility for
choosing a target sequence and provide a potential platform for protein detection in
the area of bioanalysis, disease diagnostics, and clinical biomedicine.
Acknowledgments
This work was supported by the Natural Science Foundation (NNSF) of China
(81370836)
and
the
Natural
Science
Foundation
of
Chongqing
City
(CSTC-2011JJA10078, 2010AA5041).
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17
Scheme 1
Scheme 1. Schematic illustration of the ratiometric electrochemical aptasensor.
18
Fig. 1.
Fig. 1 EIS and CV of the different modified electrodes in 0.01M PBS aqueous solution
containing 5.0 mM (1:1) [Fe(CN)6]3−/4−: (a) bare GCE, (b) Au/GCE, (c) probe/Au/GCE,
(d) HT/probe/Au/GCE, (e) HT/probe/Au/GCE incubated with Phi29, dNTPs and 5 ng
mL-1 NF-κB.
19
Fig. 2.
Fig. 2 PAGE analysis of different samples, Line 1: PD; Line2: TD; Line 3: HD; Line 4: a
mixture of HD and PD; Line 5: a mixture of PD, HD, TD, NF-κB, Phi29 and dNTPs.
20
Fig. 3.
Fig. 3 DPV curves of the different modified electrodes. (a) HT/probe/Au/GCE; (b)
HT/probe/Au/GCE incubated with Phi29 and dNTPs; (c) NF-κB/HT/probe/Au/GCE;
(d) NF-κB/HT/probe/Au/GCE incubated with Phi29 and dNTPs.
21
Fig. 4.
Fig. 4. Effects of DNA probe concentrations (A) and the time of enzyme
assisted-target protein recycling (B) on the IFc/IMB value of the aptasensor in the
presence of NF-κB (the insets show the electrochemical test results of the
optimization experiments).
22
Fig. 5.
Fig. 5. (A) DPV responses of the proposed aptasensor for the detection of different
concentrations of NF-κB. (B) logarithmic dependence of Fc (red line) and MB (black
line) peak current on target NF-κB concentration. (C) Linear relationship between
logarithm of IFc/IMB and logarithm of target NF-κB concentration.
23
Table 1. Comparisons of the proposed method with the reported method for NF-κB
detection.
Methods
EIS
Chronocoulometry
linear ranges
detection limit
reference
79 pg mL-1-158 ng mL-1
71 pg mL-1
Peng et al
0.79 ng mL-1-79 ng mL-1
0.63 ng mL-1
Ye et al
SWV
1.58 ng mL-1-79 ng mL-1
0.32 ng mL-1
Chen et al
DPV
1.58 ng mL-1 -158 ng mL-1
1.58 ng mL-1
Liang et al
0.07pg mL-1
This work
DPV
0.1pg mL-1- 15 ng mL-1
24
Table 2. Recovery results of the proposed aptasensor in human serum
Sample no.
Added/(ng mL-1)
Found/(ng mL-1)
Recovery/%
RSD/%
1
0.01
0.103
103
7.55
2
0.1
0.09
90.0
6.73
3
1.0
1.01
101
9.19
4
5.0
4.92
98.4
8.26
25
Highlights
►a simple and ingenious approach was designed to construct ratiometric aptasensor.
►the binding between NF-κB and probes induced the ratiometric signal readout.
►polymerase-assisted protein recycling amplification strategy was designed.
►the ratiometric aptasensor exhibits higher accuracy, robustness and sensitivity.
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