- Email: [email protected]
gold nanoparticles
Accepted Manuscript Label-free electrochemical aptasensor for detection of alpha-fetoprotein based on AFP-aptamer and thionin/reduced graphene oxide/gold nanoparticles Guiyin Li, Shanshan Li, Zhihong Wang, Yewei Xue, Chenyang Dong, Junxiang Zeng, Yong Huang, Jintao Liang, Zhide Zhou PII:
S0003-2697(18)30124-6
DOI:
10.1016/j.ab.2018.02.012
Reference:
YABIO 12936
To appear in:
Analytical Biochemistry
Received Date: 14 December 2017 Revised Date:
9 February 2018
Accepted Date: 12 February 2018
Please cite this article as: G. Li, S. Li, Z. Wang, Y. Xue, C. Dong, J. Zeng, Y. Huang, J. Liang, Z. Zhou, Label-free electrochemical aptasensor for detection of alpha-fetoprotein based on AFP-aptamer and thionin/reduced graphene oxide/gold nanoparticles, Analytical Biochemistry (2018), doi: 10.1016/ j.ab.2018.02.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical Abstract
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Label-free electrochemical aptasensor for detection of
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alpha-fetoprotein based on AFP-aptamer and thionin/reduced
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graphene oxide/gold nanoparticles
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Guiyin Li1, 2, Shanshan Li1, Zhihong Wang1, Yewei Xue1, Chenyang Dong1, Junxiang
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Zeng1, Yong Huang1, 2*, Jintao Liang1*, Zhide Zhou1*
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Technology, Guilin, Guangxi 541004, China
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School of Life and Environmental Sciences, Guilin University of Electronic
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Therapy, Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy
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Research, Collaborative Innovation Center for Targeting Tumor Diagnosis and
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Therapy, Guangxi Medical University, Nanning, Guangxi 530021, China
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*Corresponding author: [email protected] (Y. Huang)
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National Center for International Research of Biological Targeting Diagnosis and
[email protected] (J.T. Liang) [email protected] (Z.D. Zhou)
Tel: +86-773-2293135；Fax: +86-773-2293135
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Short title: Label-free electrochemical AFP aptasensor
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Abstract Sensitive and accurate detection of tumor markers is critical to early diagnosis,
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point-of-care and portable medical supervision. Alpha fetoprotein (AFP) is an
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important clinical tumor marker for hepatocellular carcinoma (HCC), and the
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concentration of AFP in human serum is related to the stage of HCC. In this paper, a
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label-free electrochemical aptasensor for AFP detection was fabricated using
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AFP-aptamer as the recognition molecule and thionin/reduced graphene oxide/gold
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nanoparticles (TH/RGO/Au NPs) as the sensor platform. With high electrocatalytic
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property and large specific surface area, RGO and Au NPs were employed on the
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screen-printed carbon electrode to load TH molecules. The TH not only acted as a
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bridging molecule to effectively capture and immobilize AFP-aptamer, but as the
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electron transfer mediator to provide the electrochemical signal. The AFP detection
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was based on the monitoring of the electrochemical current response change of TH by
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the differential pulse voltammetry. Under optimal conditions, the electrochemical
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responses were proportional to the AFP concentration in the range of 0.1-100.0 µg/mL.
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The limit of detection was 0.050 µg/mL at a signal-to-noise ratio of 3. The proposed
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method may provide a promising application of aptamer with the properties of facile
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procedure, low cost, high selectivity in clinic.
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Keywords: Label-free electrochemical aptasensor; Aptamer; Alpha fetoprotein;
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Thionin; Reduced graphene oxide
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1. Introduction Hepatocellular carcinoma (HCC) has been the third cause of cancer death and the
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leading cause of mortality among cirrhotic patients [1]. Alpha fetoprotein (AFP), an
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important clinical tumor marker for HCC, is a plasma protein produced by the yolk sac
5
and secreted from the liver during fetal life [2, 3]. In the serum of healthy human, AFP
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level is barely detectable with a concentration less than 25.0 ng/mL, but it increases
7
obviously to 500.0 ng/mL in nearly 75% HCC patients [4, 5]. An elevated AFP
8
concentration in adult serum is widely considered as an early indication of HCC or
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endodermal sinus tumor [6]. Therefore, the rapid, sensitive and reliable analytical
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method for AFP detection is of great significance for the early clinical diagnosis and the
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long-term treatment.
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To date, various methods have been developed for the detection of AFP, such as enzyme-linked
immunosorbent
assay
[7],
radioimmunoassay
[8],
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electrochemiluminescence [9], electrochemical immunosensor [10-13], and so on.
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Among them, electrochemical immunosensor, especially the label-free electrochemical
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immunosensor, have aroused great interests and been applied in the detection of AFP
17
owing to high sensitivity, facile operation, low cost and ease miniaturization [11, 14,
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15]. Xu et al [14] had developed a sensitive label-free immunosensor with
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anti-biofouling electrode in whole blood using anticoagulating magnetic nanoparticles
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(Fe3O4-ɛ-PL-Hep nanoparticles) for detection AFP a low detection limit of 0.072
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ng/mL. Although these studies have demonstrated that the label-free immunosensor is
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an ideal method in detection, there are still plenty of rooms for developing novel
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label-free electrochemical sensor with high performancing for AFP.
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The most crucial step for fabricating a label-free electrochemical immunosensor is
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how to immobilize the biomolecules efficiently and effectively. Until now, a variety of 3
ACCEPTED MANUSCRIPT nanomaterials have been utilized to fabricate the immunosensors, such as metal
2
nanoparticles [16, 17], carbon-based nanostructures [18], graphene sheets [19],
3
conducting polymers [20] and so on. Graphene-based nanomaterials, such as graphene
4
oxide (GO) and reduced graphene oxide (RGO), have been widely applied in
5
electrochemical sensors because of their high mobility of charge carriers, large specific
6
surface area, and upstanding electric conductivity [21, 22]. Integrating graphene with
7
gold nanoparticles (Au NPs) in constructing electrochemical biosensor, which can
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provide a better microenvironment for biomolecules’ reaction, accelerate effectively
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the electron transfer rate and amply the signal to exhibit excellent sensitivity [23, 24].
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Aptamer, screened by systematic evolution of ligands by exponential enrichment
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(SELEX), is a short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid
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(RNA) molecules [25]. Aptamer can bind to its target molecules with a high affinity
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and specificity because of its specific three-dimensional shapes. Thus, aptamer has
14
emerged as a potential candidate for biomolecular recognition in the diagnostic and
15
therapeutic fields, especially as alternative antibodies in biosensors
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Aptamer-based electrochemical sensor has been attracted increasing attention for
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clinical diagnosis and food analysis [27, 28]. In previous studies, several AFP-specific
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ssDNA and RNA aptamers were successfully screened by SELEX strategy [29-32].
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Huang et al [30] screened a AFP specific aptamer by SELEX/microfluidic chip and
20
used it to detect AFP with a linear range from 12.5 to 800 ng/mL and exhibited
21
inhibitory effects on HCC proliferation. Dong et al [31] selected an AFP-specific
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ssDNA aptamer, named AP273, based on SELEX/ capillary electrophoresis, and the
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AFP specific aptamers could be used potentially as a novel diagnostic and therapeutic
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agent in AFP positive HCC patients.
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[26].
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In this paper, a simple label-free electrochemical aptasensor for AFP detection has 4
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using
thionin/reduced
graphene
oxide/gold
nanoparticles
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(TH/RGO/Au NPs) as the immobilization platform for capture the AFP-aptamer. After
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the AFP-aptamer quickly recognized AFP via specific recognition reaction, the
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electrochemical response signal of the aptasensor was recorded by differential pulse
5
voltammetry (DPV). The characterization and analytical performance of the prepared
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aptasensor were studied in detail. This strategy could be further developed for practical
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clinical detection of AFP using aptamer instead of antibody.
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2. Materials and methods
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2.1. Chemicals and reagents
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Alpha fetoprotein (AFP), immunoglobulin G (Ig G) and immunoglobulin E (Ig E)
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were purchased from Shanghai Linc-Bio Science Co., Ltd (Shanghai, China). Bovine
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serum albumin (BSA), Human serum albumin (HSA) and HAuCl4 were supplied by
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Sangon Biotech (Shanghai, China). Graphene oxide (GO) was purchased from Nanjing
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Jcnano Tech Co., Ltd (Nanjing, China). Thionin (TH) was provided by Shanghai Regal
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Biology Tech Co., Ltd (Shanghai, China). Tris(Hydroxymethyl)aminomethane (Tris)
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was obtained from Beijing Baishayi Tech Co., Ltd (Beijing, China). Clinical human
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serum sample was kindly provided by the 181st Hospital of Chinese People’s
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Liberation Army (Guilin, China). All other reagents were of analytical grade and used
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without any further purification. All solutions were prepared with ultrapure water of 18
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MΩ·cm purified from a Milli-Q purification system (Milli-Pore, Bedford, MA, USA).
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The AFP-aptamer with the following sequences [31] was purchased from Sangon
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biotech Co., Ltd (Shanghai, China). 5'-GTGACGCTCCTAACGCTGACTCAGGTGCAGTTCTCGACTCGGTCTT GATGTGGGTCCTGTCCGTCCGAACCAATC-3' Tris-HCl buffer solution (0.1 mol/L, pH 7.0) was used to prepare different 5
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concentration of AFP-aptamer solution.
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2.2. Apparatus The prepared aptasensor was characterized by scanning electron microscopy
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(SEM, Quanta 200, Elementar, Germany), Raman microscope (Thermo DXRXi,
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Renishaw, UK) and X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi,
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ThermoFisher, USA). All electrochemical measurements were performed on a CHI660
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electrochemical workstation (Shanghai Chenhua Instrument, China) at room
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temperature. A conventional screen-printed electrode system (SPE, Nanjing Yunyou
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Biotech Co., Ltd, China) was used for all electrochemical measurements: one carbon
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electrode as the working electrode, another carbon electrode as the auxiliary electrode
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and Ag/AgCl electrode as the reference electrode. Cyclic voltammetry (CV) was
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performed in phosphate buffer saline (PBS, pH 7.0, 0.1 mol/L Na2HPO4/KH2PO4 and
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0.1 mol/L NaCl) with scanning range from -1.0 to 1.0 V and scanning rate of 100 mV/s.
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Electrochemical impedance spectroscopy (EIS) was acquired in 10.0 mmol/L
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K3Fe(CN)6/K4Fe(CN)6 containing 0.1 mol/L KCl solutions at 0.24 V from 1 to 100
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KHz.
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2.3. Fabrication of the label-free electrochemical aptasensor
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The RGO was prepared according to a previous paper with some modifications
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[33]. Briefly, 10.0 mL of 0.1 mg/mL of GO was dispersed with ultrasonic for 2 h to
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form a uniform suspension, then 10 mg ascorbic acid (AA) was slowly added and
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stirred for 12 h. Following that, the mixed solution was centrifuged for 15 min at
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10000 rpm, removed the supernatant, washed twice with ultrapure water and got the
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RGO.
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Prior to the experiment, the bare SPE was pretreated in 0.5 mol/L H2SO4 by CV
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method for 10 cycles with the scanning voltage of -0.4 -1.2 V and the scanning rate of 6
ACCEPTED MANUSCRIPT 100 mV/s. Then, the pretreated SPE was immersed in 5.0 mL of 0.01% HAuCl4
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solution at room temperature (RT) and electrodeposited Au NPs on the surface of SPE
3
at a potential of -0.5 V for 120s [34]. After the electrode was cleaned with water and
4
dried at RT, 3.0 µL of 0.1 mg/mL RGO solution was dropped on the surface of Au
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NPs/SPE, irradiated in the infrared light for 30 min. Afterwards, 2.5 µL of 10 mmol/L
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TH was added to the surface of RGO/Au NPs/SPE and dried at RT. Following that,
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3.0 µL of 5.0 µmol/L AFP-aptamer solution was dropped on the surface of
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TH/RGO/Au NPs/SPE and incubated for 2 h at RT, and then washed twice with PBS
9
(0.1 mol/L, pH 7.0) to remove the excess aptamer. Finally, 0.5% BSA was used to
10
block the possible remaining active sites against nonspecific adsorption and rinsed
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with PBS. Thus, the label-free electrochemical aptasensor (AFP-aptamer/TH/RGO/Au
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NPs/SPE) was fabricated, and stored in a refrigerator at 4 ºC before use.
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2.4. Electrochemical measurement the AFP concentration with the label-free
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electrochemical aptasensor
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To carry out the electrochemical measurement, 3.0 µL of different AFP
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concentration was dropped onto the surface of AFP-aptamer/ TH/RGO/Au NPs/SPE.
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After incubated for 60 min at 25 ºC, the electrode was washed twice with PBS. Then,
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differential pulse voltammetry (DPV) was performed in PBS (0.2 mol/L, pH 6.5)
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from -0.7 to -0.25 V to record the peak currents of the aptasensor for quantitative
20
analysis. Each sample was detected for three times and the results were calculated as
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mean±RSD.
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3. Result and discussion
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3.1. Analytical principle of the label-free electrochemical aptasensor for AFP
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detection
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ACCEPTED MANUSCRIPT TH/RGO/Au NPs/SPE for AFP detection, and the analytical principle was illustrated in
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Fig. 1A. Firstly, a layer of Au NPs was deposited on the surface of the SPE by
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electrodeposition. Secondly, RGO was loaded on the surface of Au NPs/SPE due to the
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electrostatic adsorption. Following that, TH was assembled to the RGO/Au NPs/SPE
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through electrostatic interactions. Then, AFP-aptamer was immobilized on the surface
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of TH/RGO/Au NPs/SPE by electrostatic adsorption due to TH containing amino
7
groups and easy to intercalate with nucleic acid molecules [35, 36]. When AFP
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solution was dropped onto the surface of AFP-aptamer/TH/RGO/Au NPs/SPE, the
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AFP-aptamer quickly recognized AFP via specific recognition reaction, which made
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the change of electrochemical signals of TH obtained by DPV. The value of
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electrochemical signal increased accordingly with the increase of the concentration of
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AFP. Thus, a high sensitivity of the label-free electrochemical aptasensor could be
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achieved.
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Herein, TH/RGO/Au NPs was employed as a nanocarrier to load AFP-aptamer.
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The decorated TH on the electrode acted as not only a bridging molecule to effectively
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capture and immobilize AFP-aptamer, but also the signal indicator for monitoring the
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concentration of the AFP. The electrochemical signal of TH could be amplified
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effectively by the synergistic effect between Au NPs with good electrocatalytic
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activity and RGO with high specific surface area.
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Fig. 1B illustrated the feasibility of the developed aptasensor for AFP detection
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using the DPV method. Seen from Fig. 1B, there was a remarkable current response
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(67.00 µA) at -344 mV in the presence of AFP (curve a). As a control, there appeared a
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mild current response (36.00 µA) at -348 mV without AFP (curve b). This may be due
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to the following reasons: The AFP-aptamer firstly covered the electrode surface which
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may hinder the electron transfer and the current was mild. When AFP solution was 8
ACCEPTED MANUSCRIPT dropped onto the surface of AFP-aptamer/TH/RGO/Au NPs/SPE, the AFP-aptamer
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was binding with AFP molecules via specific recognition reaction, and formed the
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aptamer-antigen complexes. The produced complexes were arranged with stable spatial
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structure on the surface of the electrode, providing more space for electron transfer,
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thus the current increased.
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3.2. Electrochemical characterization of the label-free electrochemical aptasensor
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In order to monitor the changes of electrochemical behavior at different stages of
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the aptasensor, CV was performed in PBS (pH 7.0, 0.1 mol/L Na2HPO4/KH2PO4 and
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0.1 mol/L NaCl) with scanning range from -1.0 to 1.0 V and scanning rate of 100 mV/s
11
and the results were shown in Fig. 1C. The bare SPE (curve a) had no significant redox
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peaks current without electroactive material in the electrode. After the Au NPs was
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electro-deposited on the surface of SPE, an obvious cathodic peak appeared (curve b)
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because of the highly conductivity of Au NPs. Compared to curve b, the cathodic peak
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current was slightly decreased when the RGO was integrated on the surface of Au
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NPs/SPE (curve c), owing to the formation of a thin RGO film on the electrode.
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Subsequently, the reversible reduction and oxidation peaks with cathodic and anodic
18
peak potentials of TH were observed (curve d), which indicating that TH could served
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as an electrochemical indicator for detection. After incubation with the AFP-aptamer,
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an obvious decrease of the peak current was observed (curve e), this may be the large
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resistance of the aptamer blocked the ability of electron transfer of TH at the sensing
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interface. The results also indicated that the electrostatic interaction between TH and
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Au NPs was very strong, and that AFP-aptamer could be firmly immobilized on the
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electrode surface. Finally, the cathodic and anodic peaks’ current obviously increased
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(curve
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f)
when
AFP
solution
was
dropped
onto
the
surface
of 9
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AFP-aptamer/TH/RGO/Au NPs/SPE. Since the specific recognition between
2
aptamer-antigen which provided the space for electron transfer and accelerated the
3
electron transfer rate. What’s more, electrochemical impedance spectroscopy (EIS), a valid method for
5
exploring the properties of the surface of the modified electrodes, was performed at
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0.24V in 10.0 mmol/L K3Fe(CN)6/K4Fe(CN)6 containing 0.1 mol/L KCl solutions at
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the frequency range from 1 to 100 KHz, and the results were shown in Fig. 2A. Seen
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from Fig. 2A, a large semicircle can be observed for the bare SPE (curve a), which
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implied that the impedance of the bare SPE was relatively high and displayed low Ret
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(electron transfer resistance). When Au NPs was deposited on the SPE, the Ret
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decreased obviously (curve b), implying that the Au NPs could greatly accelerate the
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electron transfer. After RGO and TH were modified on the surface of Au NPs/SPE, the
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semicircle further reduced (curve c and curve d), due to the upstanding electric
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conductivity of RGO and TH. Compared to the curve d, the Ret increased remarkably
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after the AFP-aptamer was immobilized (curve e), and decreased again when the
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specific recognition reaction between aptamer-antigen occurred (curve f). The EIS
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results were in good agreement with those obtained from CV, further revealing the
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successful fabrication of the label-free electrochemical aptasensor.
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3.3. Raman characterization of the label-free electrochemical aptasensor Fig. 2B depicted the Raman spectroscopy for the different stages of the electrode
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modification. As can be seen from Fig. 2B, there are two main characteristic Raman
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bands in 1350 cm-1 (D-band) and 1580 cm-1 (G-band), respectively. Compared to the
24
curve b, the signal strength of curve c was significantly enhanced. By calculation, the
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value of ID/IG for RGO/Au NPs/SPE was 0.97, indicating that RGO successfully fixed 10
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on the electrode. After fixed TH and AFP-aptamer, the intensity of Raman bands did
2
not change much, implying that RGO did not fell down from the surface of electrode.
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3.4. SEM characterization of the label-free electrochemical aptasensor Fig. 2C-2H demonstrated SEM images of the process of the label-free
5
electrochemical aptasensor at different stages. In Fig. 2C, the bare SPE gave a uniform
6
dark image without particles on the surface. There were homogeneously distributed
7
spherical particles with the diameter of 80 ± 10 nm in the image of Au NPs/SPE (Fig.
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2D), indicating the successful deposition of Au NPs onto the surface of SPE. Fig. 2E
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depicted the SEM image of the RGO/Au NPs/SPE. The white spherical particles
10
became smaller and blurred compared to the image of Au NPs/SPE, since the RGO
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was present on the surface of the Au NPs/SPE with a thin film. In addition, a substance
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similar to the shape of the branches could be clearly observed on the surface of
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TH/RGO/Au NPs/SPE (Fig. 2F), implying that TH binded with RGO and Au NPs
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firmly. When the AFP-aptamer was immobilized on the surface of TH/RGO/Au
15
NPs/SPE, no apparent structure was seen in Fig. 2G. Fig.2H was the SEM image of the
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SPE after AFP added. Because of the smaller particle size of AFP molecules, the
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difference between Fig. 2G and Fig. 2H was not significant.
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3.5. XPS characterization of the label-free electrochemical aptasensor
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To further analyze the fabrication process of the aptasensor, X-ray photoelectron
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spectroscopy (XPS) measurement was performed. Four main features including O1s,
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N1s, C1s and Au4f peaks of the XPS spectrum were inspected (Fig. 3). Seen from Fig.
22
3, Compared curve a with curve b, the Au4f (82.70 eV) signal increased sharply, while
23
C1s (283.40 eV) signal decreased slightly due to the electrolytic deposition of Au NPs.
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After RGO was dropped onto the surface of Au NPs/SPE, the signal of C1s (283.20 eV)
25
rised and Au4f (82.70 eV) signal decreased (curve c). However, the further decrease of 11
ACCEPTED MANUSCRIPT Au4f (82.20 eV) peaks accompanied with the increase in the peak intensities of N1s
2
(397.70 eV) and O1s (530.70 eV) appeared with TH decoration (curve d),
3
demonstrating that TH has been successfully attached to the surface of RGO/Au
4
NPs/SPE. After immobilization of AFP-aptamer and AFP, the total intensity of O1s
5
(531.00 eV) and N1s (398.00 eV) increased continuously, which promoted the decrease
6
of C1s (284.00 eV) signal and the disappearance of Au4f (83.00 eV) signal (curve e and
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curve f). The XPS data fully confirmed that the biological molecules were correctly
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immobilized on the electrode surface.
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A further insight can be obtained from the inspection of table inserted in Fig. 3A,
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which summarized the relative abundance (% of atoms) of the various elements (O, N,
11
C, Au). From a to f, a strong increase in the proportion of O from 16.56% to 38.49%
12
and a bit increase in the proportion of N from 8.06% to 11.33% while a mild decrease
13
in the proportion of C from 75.38% to 50.18%. In addition, the proportion of Au
14
decreased strongly from 8.30% to 0.08%.
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3.6. Optimization of experimental conditions for detection AFP using the
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label-free electrochemical aptasensor
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To achieve the best results for the sensor, the experimental conditions, such as the
19
incubation time, the pH of the buffer solution, incubation temperature and the
20
concentration of AFP aptamer, were optimized. Fig. 4A showed the effect of the
21
incubation time on the current response of the aptasensor. Seen from Fig. 4A, the
22
current response value increased (from 56.69 µA to 61.65 µA) with the increase of the
23
incubation time from 15 min to 30 min, and reached maximum (71.99 µA) when the
24
incubation time was 60 min. When the incubation time exceeded to 60 min, the current
25
response value tended to decrease (56.44 µA). Therefore, the incubation time was
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selected for 60 min for the further experiment. Fig. 4B depicted the influences of the incubation temperature on the current
3
response of the aptasensor. It has been known that the higher and lower temperature
4
were all harmful to the biomolecule activity. The response value of the biosensor
5
increased from 43.97 µA to 72.00 µA with the incubation temperature increase from 4
6
to 25 ºC. When the temperature was 25 ºC, the corresponding current reached the
7
maximum (72.00 µA). As the temperature continued to rise, the response current
8
decreased. Therefore, 25 ºC was selected for the optimal temperature.
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The pH of the buffer solution is a vital parameter for the aptasensor because the
10
activity of the immobilized protein could be influenced by the acidity of the solution.
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So the aptasensor was tested in a series of PBS solution with the pH values varying
12
from 5.7 to 8.0. As shown in Fig. 4C, with increasing pH value from 5.7 to 6.5, the
13
current responses increased (from 50.87 µA to 72.54 µA), and then the value decreased
14
(61.67 µA, 48.63 µA, 45.45 µA) rapidly at the higher pH values (7.0, 7.5, 8.0). As a
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consequence, the optimal pH 6.5 was used for further experiment.
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The effect of the concentration of AFP-aptamer on the current response of the
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aptasensor was shown in Fig. 4D. It can be clearly observed that the current response
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increased from 50.18 µA to 71.41 µA with increasing concentration of AFP-aptamer
19
and reached a maximum (71.41 µA) at 5.0 µmol/L, implying that AFP-aptamer and
20
AFP reacted completely. Due to the excess aptamer could hinder electron transfer,
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higher AFP-aptamer concentration made the response decrease. Therefore, 5.0 µmol/L
22
was selected as the optimum concentration of AFP-aptamer.
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ACCEPTED MANUSCRIPT standard solutions using the DPV method. Under the optimized conditions, the
2
developed aptasensor was used to detect different concentrations of AFP and the
3
current response was recorded. Fig. 5A showed the electrochemical signal responses
4
of the proposed aptasensor for the detection of AFP concentration in the range of
5
0.1-100.0 µg/mL. Seen from Fig. 5A, the current response of the aptasensor increased
6
along with the increasing concentration of AFP, which confirmed the formation
7
between the AFP and the AFP-aptamer. As the concentration of AFP increasing, a
8
larger amount of AFP was specifically recognized by AFP-aptamer, leading to the
9
gradually increasing electrochemical signal response. As can be seen from Fig. 5B, the
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current response was linearly proportional with the AFP concentration in the range of
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0.1 - 100.0 µg/mL. The regression equation was Y = 0.3471X + 47.984 (Y was the peak
12
current and X indicated the concentration of AFP) with a correlation coefficient of
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0.9987. Specifically, the limit of detection (LOD) can be calculated at a signal/noise of
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3 based on the standard deviation of the response (σ) and the slope (B) of the calibration
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curve [37, 38]. According to the formula: LOD = 3(σ/B), LOD was determined to be
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0.05 µg/mL (where σ was the standard deviation in blank sample, n=3). Compared to
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other reported AFP immunosensors [11, 14, 39, 40], the proposed aptasensor showed a
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high detection limit, which may be the following reason: The affinity between
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AFP-aptamer and AFP was not strong enough and the reaction was not complete; In
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addition, the aptamer-antigen immunocomplexs had spatial stereoscopic effect and
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hindered electron transfer to make decrease of current change.
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Although the proposed aptasensor showed a high detection limit and can not fully
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meet the clinical requirements at present, the simple and low-cost of aptamer provided
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a new promising platform for the design of the highly sensitive detection method in
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clinical immunoassays. 14
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3.8. Reproducibility, specificity and stability of the label-free electrochemical
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aptasensor To evaluate the reproducibility of the aptasensor, five freshly prepared modified
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electrodes were employed for the detection of AFP (100.0 µg/mL). All five electrodes
6
exhibited similar current response (54.96 µA, 54.72 µA, 48.93 µA, 59.55 µA, 52.44 µA)
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and the inter-assay relative standard deviation (RSD) was 3.9%, suggesting that the
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proposed aptasensor had quite good reproducibility for AFP detection.
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Moreover, some biological molecules, such as BSA, HSA, IgG and IgE, were
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chosen as interferences to evaluate the specificity of the aptasensor. Fig. 5C showed the
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DPV current response of the aptasensor to all the above interferences (100.0 µg/mL)
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instead of the AFP under the optimal conditions. Seen from Fig. 5C, when the test
13
sample was BSA, HSA, IgG and IgE, the aptasensor's current response value were
14
40.02 µA, 38.38 µA, 33.22 µA and 32.94 µA, respectively, accounting for 48.19%,
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45.78%, 39.75%, 38.55% of the current response value of AFP (82.93 µA),
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respectively. The result showed that the aptasensor exhibits good specificity for AFP.
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Additionally, the stability of the biosensor was studied after storage at 4 ºC for 7
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and 14 days, respectively. The current response were 78.73 µA and 74.23 µA. The
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activity of the immunosensor can be maintained at 95.76% and 90.86%, demonstrating
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good stability of the aptasensor.
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3.9. Real sample analysis
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In order to verify the practical application of the fabricated sensor, the feasibility
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of the proposed method for the detection of human serum was evaluated by standard
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addition method. The human serum sample was donated by the local hospital, and the
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level of AFP in this sample was calculated to be 10.6 ng/mL by ELISA. 3 µL of known 15
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surface of AFP-aptamer/TH/RGO/Au NPs/SPE. Then, 100.0 µL of human serum
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sample were added to 5 mL of PBS solution (0.2 mol/L, pH 6.5), the levels of AFP in
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spiked samples were parallelly assayed for 3 times with DPV using the developed
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aptasensor. The experimental results were shown in Table 1. Results can be seen from
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Table 1 that the recoveries were in the range from 101.52% to 107.95%. It can be
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known that the recovery results were high which could meet the needs of the actual
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sample detection.
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In summary, a label-free electrochemical aptasensor for the detection of AFP
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using TH/RGO/AuNPs as the immobilization platform and AFP aptamer as the
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recognition molecule has been successfully developed. Combining Au NPs (high
14
conductivity and biocompatibility), RGO (large specific surface area and upstanding
15
electric conductivity), TH (electrochemical indicator) and AFP aptamer (high affinity
16
and specificity), the proposed aptasensor showed excellent performance with highly
17
selective, simple operation and long-term stability. In the range of 0.1 to 100.0 µg/mL,
18
a linear curve was obtained (Y=0.3471X + 47.984) between current and AFP
19
concentrations with a readily achievable detection limit of 0.050 µg/mL at a
20
signal/noise ratio of 3. Although the proposed aptasensor showed a high detection limit
21
and can not fully meet the clinical requirements at present. It was worth pondering that
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we need to further optimize the sensor preparation process to improve the detection line.
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Most importantly, the simple and cost-effective sensing strategy provides a new
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promising platform for the design of the highly sensitive detection method, showing
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potential application for aptamer in clinical immunoassays.
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Acknowledgments This work was supported by the National Nature Science Foundation of China
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(Nos. 81460451, 81760534 and 81430055), the Innovation Project of GUET Graduate
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Education (2017YJCX95, YCSW2017146), the Appropriate Health Technology
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Development Project of Guangxi Zhuang Autonomous Region (No. S201422-03), the
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National
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2015GXNSFDA139025, 2016GXNSFAA380011 and 2016GXNSFAA380080) and
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the Foundation of Guangxi Key Laboratory of Automatic Detecting Technology and
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Instruments (No. YQ17114).
of
Guangxi
province
of
China
(Nos.
SC
Foundation
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ACCEPTED MANUSCRIPT Figure captions
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Fig. 1 (A) Principle of the label-free electrochemical aptasensor for detection AFP
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based on AFP-aptamer and TH/RGO/Au NPs. (B) DPV response of the label-free
4
electrochemical aptasensor with AFP (a) and without AFP (b). The concentration of
5
AFP was 100.0 µg/mL and the DPV signal was obtained in PBS (0.2 mol/L, pH 6.5)
6
from -0.7 to -0.25 V. (C) CV response of (a) bare SPE, (b) Au NPs/SPE, (c) RGO/Au
7
NPs/SPE, (d) TH/RGO/Au NPs/SPE, (e) AFP-aptamer/TH/RGO/Au NPs/SPE, (f)
8
AFP/AFP-aptamer/TH/RGO/Au NPs/SPE.
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Fig. 2 (A) Electrochemical impedance spectroscopy of (a) bare SPE, (b) Au NPs/SPE,
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(c) RGO/Au NPs/SPE, (d) TH/RGO/Au NPs/SPE, (e) AFP-aptamer/TH/RGO/Au
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NPs/SPE, (f) AFP/AFP-aptamer/TH/RGO/Au NPs/SPE. (B) Raman spectra of (a) bare
12
SPE, (b) Au NPs/SPE, (c) RGO/Au NPs/SPE, (d) TH/RGO/Au NPs/SPE, (e)
13
AFP-aptamer/TH/RGO/Au NPs/SPE, (f) AFP/AFP-aptamer/TH/RGO/Au NPs/SPE.
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(C) SEM image of bare SPE. (D) SEM image of Au NPs/SPE. (E) SEM image of
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RGO/Au NPs/SPE. (F) SEM image of TH/RGO/Au NPs/SPE. (G) SEM image of
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AFP-aptamer/TH/RGO/Au
17
AFP/AFP-aptamer/TH/RGO/Au NPs/SPE.
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Fig. 3 (A) Full XPS spectra of the surface of SPE in different stages. (B) XPS spectra of
19
C1s, O1s, N1s and Au4f. Among them (a) bare SPE, (b) Au NPs/SPE, (c) RGO/Au
20
NPs/SPE, (d) TH/RGO/Au NPs/SPE, (e) AFP-aptamer/TH/RGO/Au NPs/SPE, (f)
21
AFP/AFP-aptamer/TH/RGO/Au NPs/SPE.
22
Fig. 4 (A) Effect of incubation time on the current signals. (B) Effect of incubation
23
temperature on the current signals. (C) Effect of pH on the current signals. (D) Effect of
24
the concentration of aptamer on the current signals. The concentration of AFP was
25
100.0 µg/mL and the DPV current signal was obtained in PBS solution (0.2 mol/L, pH
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(H)
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ACCEPTED MANUSCRIPT 6.5) from -0.7 to -0.25 V with a 100 mV/s scanning rate. And the error bars represent
2
the relative standard deviation (RSD) (n=3 electrodes).
3
Fig. 5 (A) Electrochemical signal responses of the label-free electrochemical
4
aptasensor for the detection of different concentrations of AFP including 100.0, 80.0,
5
65.0, 50.0, 40.0, 25.0, 10.0, 1.0 and 0.1µg/mL. (B) Calibration curve of the aptasensor
6
for the detection of different concentrations of AFP. (C) Specificity of the proposed
7
aptasensor with BSA, HSA, IgG and IgE instead of AFP. The current signal was
8
obtained by DPV which was carried out in PBS (0.2 mol/L, pH 6.5) from -0.7 to -0.25
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V with a 100 mV/s scanning rate. And the error bars represented the relative standard
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Tables
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Table 1 Determination of AFP by the proposed aptasensor in PBS (0.2 mol/L, pH 6.5)
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containing healthy human serum from -0.7 to -0.25 V with a 100 mV/s scanning rate
AFP Added
Averange
(ng/mL)
(µg/mL)
(µg/mL)
15.00
16.12
20.00
21.59
50.00
50.76
Recovery
(%)
(%)
1.13
107.47%
0.36
107.95%
3.47
101.52%
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AFP in samples
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ACCEPTED MANUSCRIPT Hightlights A simple label-free electrochemical aptasensor based on TH/RGO/Au NPs was developed.
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TH/RGO/Au NPs was the immobilization platform for capture the AFP-aptamer.
The electrochemical responses were proportional to the AFP concentration.
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The proposed aptasensor showed facile procedure, low cost and high
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selectivity.
S0003-2697(18)30124-6
DOI:
10.1016/j.ab.2018.02.012
Reference:
YABIO 12936
To appear in:
Analytical Biochemistry
Received Date: 14 December 2017 Revised Date:
9 February 2018
Accepted Date: 12 February 2018
Please cite this article as: G. Li, S. Li, Z. Wang, Y. Xue, C. Dong, J. Zeng, Y. Huang, J. Liang, Z. Zhou, Label-free electrochemical aptasensor for detection of alpha-fetoprotein based on AFP-aptamer and thionin/reduced graphene oxide/gold nanoparticles, Analytical Biochemistry (2018), doi: 10.1016/ j.ab.2018.02.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical Abstract
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Label-free electrochemical aptasensor for detection of
2
alpha-fetoprotein based on AFP-aptamer and thionin/reduced
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graphene oxide/gold nanoparticles
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Guiyin Li1, 2, Shanshan Li1, Zhihong Wang1, Yewei Xue1, Chenyang Dong1, Junxiang
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Zeng1, Yong Huang1, 2*, Jintao Liang1*, Zhide Zhou1*
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1
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Technology, Guilin, Guangxi 541004, China
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School of Life and Environmental Sciences, Guilin University of Electronic
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2
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Therapy, Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy
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Research, Collaborative Innovation Center for Targeting Tumor Diagnosis and
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Therapy, Guangxi Medical University, Nanning, Guangxi 530021, China
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*Corresponding author: [email protected] (Y. Huang)
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National Center for International Research of Biological Targeting Diagnosis and
[email protected] (J.T. Liang) [email protected] (Z.D. Zhou)
Tel: +86-773-2293135；Fax: +86-773-2293135
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Short title: Label-free electrochemical AFP aptasensor
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Abstract Sensitive and accurate detection of tumor markers is critical to early diagnosis,
3
point-of-care and portable medical supervision. Alpha fetoprotein (AFP) is an
4
important clinical tumor marker for hepatocellular carcinoma (HCC), and the
5
concentration of AFP in human serum is related to the stage of HCC. In this paper, a
6
label-free electrochemical aptasensor for AFP detection was fabricated using
7
AFP-aptamer as the recognition molecule and thionin/reduced graphene oxide/gold
8
nanoparticles (TH/RGO/Au NPs) as the sensor platform. With high electrocatalytic
9
property and large specific surface area, RGO and Au NPs were employed on the
10
screen-printed carbon electrode to load TH molecules. The TH not only acted as a
11
bridging molecule to effectively capture and immobilize AFP-aptamer, but as the
12
electron transfer mediator to provide the electrochemical signal. The AFP detection
13
was based on the monitoring of the electrochemical current response change of TH by
14
the differential pulse voltammetry. Under optimal conditions, the electrochemical
15
responses were proportional to the AFP concentration in the range of 0.1-100.0 µg/mL.
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The limit of detection was 0.050 µg/mL at a signal-to-noise ratio of 3. The proposed
17
method may provide a promising application of aptamer with the properties of facile
18
procedure, low cost, high selectivity in clinic.
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Keywords: Label-free electrochemical aptasensor; Aptamer; Alpha fetoprotein;
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Thionin; Reduced graphene oxide
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1. Introduction Hepatocellular carcinoma (HCC) has been the third cause of cancer death and the
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leading cause of mortality among cirrhotic patients [1]. Alpha fetoprotein (AFP), an
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important clinical tumor marker for HCC, is a plasma protein produced by the yolk sac
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and secreted from the liver during fetal life [2, 3]. In the serum of healthy human, AFP
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level is barely detectable with a concentration less than 25.0 ng/mL, but it increases
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obviously to 500.0 ng/mL in nearly 75% HCC patients [4, 5]. An elevated AFP
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concentration in adult serum is widely considered as an early indication of HCC or
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endodermal sinus tumor [6]. Therefore, the rapid, sensitive and reliable analytical
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method for AFP detection is of great significance for the early clinical diagnosis and the
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long-term treatment.
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To date, various methods have been developed for the detection of AFP, such as enzyme-linked
immunosorbent
assay
[7],
radioimmunoassay
[8],
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electrochemiluminescence [9], electrochemical immunosensor [10-13], and so on.
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Among them, electrochemical immunosensor, especially the label-free electrochemical
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immunosensor, have aroused great interests and been applied in the detection of AFP
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owing to high sensitivity, facile operation, low cost and ease miniaturization [11, 14,
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15]. Xu et al [14] had developed a sensitive label-free immunosensor with
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anti-biofouling electrode in whole blood using anticoagulating magnetic nanoparticles
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(Fe3O4-ɛ-PL-Hep nanoparticles) for detection AFP a low detection limit of 0.072
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ng/mL. Although these studies have demonstrated that the label-free immunosensor is
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an ideal method in detection, there are still plenty of rooms for developing novel
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label-free electrochemical sensor with high performancing for AFP.
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The most crucial step for fabricating a label-free electrochemical immunosensor is
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how to immobilize the biomolecules efficiently and effectively. Until now, a variety of 3
ACCEPTED MANUSCRIPT nanomaterials have been utilized to fabricate the immunosensors, such as metal
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nanoparticles [16, 17], carbon-based nanostructures [18], graphene sheets [19],
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conducting polymers [20] and so on. Graphene-based nanomaterials, such as graphene
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oxide (GO) and reduced graphene oxide (RGO), have been widely applied in
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electrochemical sensors because of their high mobility of charge carriers, large specific
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surface area, and upstanding electric conductivity [21, 22]. Integrating graphene with
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gold nanoparticles (Au NPs) in constructing electrochemical biosensor, which can
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provide a better microenvironment for biomolecules’ reaction, accelerate effectively
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the electron transfer rate and amply the signal to exhibit excellent sensitivity [23, 24].
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Aptamer, screened by systematic evolution of ligands by exponential enrichment
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(SELEX), is a short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid
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(RNA) molecules [25]. Aptamer can bind to its target molecules with a high affinity
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and specificity because of its specific three-dimensional shapes. Thus, aptamer has
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emerged as a potential candidate for biomolecular recognition in the diagnostic and
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therapeutic fields, especially as alternative antibodies in biosensors
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Aptamer-based electrochemical sensor has been attracted increasing attention for
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clinical diagnosis and food analysis [27, 28]. In previous studies, several AFP-specific
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ssDNA and RNA aptamers were successfully screened by SELEX strategy [29-32].
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Huang et al [30] screened a AFP specific aptamer by SELEX/microfluidic chip and
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used it to detect AFP with a linear range from 12.5 to 800 ng/mL and exhibited
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inhibitory effects on HCC proliferation. Dong et al [31] selected an AFP-specific
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ssDNA aptamer, named AP273, based on SELEX/ capillary electrophoresis, and the
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AFP specific aptamers could be used potentially as a novel diagnostic and therapeutic
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agent in AFP positive HCC patients.
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[26].
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In this paper, a simple label-free electrochemical aptasensor for AFP detection has 4
ACCEPTED MANUSCRIPT been
using
thionin/reduced
graphene
oxide/gold
nanoparticles
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(TH/RGO/Au NPs) as the immobilization platform for capture the AFP-aptamer. After
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the AFP-aptamer quickly recognized AFP via specific recognition reaction, the
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electrochemical response signal of the aptasensor was recorded by differential pulse
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voltammetry (DPV). The characterization and analytical performance of the prepared
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aptasensor were studied in detail. This strategy could be further developed for practical
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clinical detection of AFP using aptamer instead of antibody.
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2. Materials and methods
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2.1. Chemicals and reagents
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Alpha fetoprotein (AFP), immunoglobulin G (Ig G) and immunoglobulin E (Ig E)
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were purchased from Shanghai Linc-Bio Science Co., Ltd (Shanghai, China). Bovine
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serum albumin (BSA), Human serum albumin (HSA) and HAuCl4 were supplied by
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Sangon Biotech (Shanghai, China). Graphene oxide (GO) was purchased from Nanjing
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Jcnano Tech Co., Ltd (Nanjing, China). Thionin (TH) was provided by Shanghai Regal
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Biology Tech Co., Ltd (Shanghai, China). Tris(Hydroxymethyl)aminomethane (Tris)
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was obtained from Beijing Baishayi Tech Co., Ltd (Beijing, China). Clinical human
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serum sample was kindly provided by the 181st Hospital of Chinese People’s
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Liberation Army (Guilin, China). All other reagents were of analytical grade and used
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without any further purification. All solutions were prepared with ultrapure water of 18
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MΩ·cm purified from a Milli-Q purification system (Milli-Pore, Bedford, MA, USA).
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The AFP-aptamer with the following sequences [31] was purchased from Sangon
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biotech Co., Ltd (Shanghai, China). 5'-GTGACGCTCCTAACGCTGACTCAGGTGCAGTTCTCGACTCGGTCTT GATGTGGGTCCTGTCCGTCCGAACCAATC-3' Tris-HCl buffer solution (0.1 mol/L, pH 7.0) was used to prepare different 5
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concentration of AFP-aptamer solution.
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2.2. Apparatus The prepared aptasensor was characterized by scanning electron microscopy
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(SEM, Quanta 200, Elementar, Germany), Raman microscope (Thermo DXRXi,
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Renishaw, UK) and X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi,
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ThermoFisher, USA). All electrochemical measurements were performed on a CHI660
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electrochemical workstation (Shanghai Chenhua Instrument, China) at room
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temperature. A conventional screen-printed electrode system (SPE, Nanjing Yunyou
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Biotech Co., Ltd, China) was used for all electrochemical measurements: one carbon
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electrode as the working electrode, another carbon electrode as the auxiliary electrode
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and Ag/AgCl electrode as the reference electrode. Cyclic voltammetry (CV) was
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performed in phosphate buffer saline (PBS, pH 7.0, 0.1 mol/L Na2HPO4/KH2PO4 and
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0.1 mol/L NaCl) with scanning range from -1.0 to 1.0 V and scanning rate of 100 mV/s.
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Electrochemical impedance spectroscopy (EIS) was acquired in 10.0 mmol/L
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K3Fe(CN)6/K4Fe(CN)6 containing 0.1 mol/L KCl solutions at 0.24 V from 1 to 100
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KHz.
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2.3. Fabrication of the label-free electrochemical aptasensor
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The RGO was prepared according to a previous paper with some modifications
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[33]. Briefly, 10.0 mL of 0.1 mg/mL of GO was dispersed with ultrasonic for 2 h to
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form a uniform suspension, then 10 mg ascorbic acid (AA) was slowly added and
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stirred for 12 h. Following that, the mixed solution was centrifuged for 15 min at
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10000 rpm, removed the supernatant, washed twice with ultrapure water and got the
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RGO.
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Prior to the experiment, the bare SPE was pretreated in 0.5 mol/L H2SO4 by CV
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method for 10 cycles with the scanning voltage of -0.4 -1.2 V and the scanning rate of 6
ACCEPTED MANUSCRIPT 100 mV/s. Then, the pretreated SPE was immersed in 5.0 mL of 0.01% HAuCl4
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solution at room temperature (RT) and electrodeposited Au NPs on the surface of SPE
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at a potential of -0.5 V for 120s [34]. After the electrode was cleaned with water and
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dried at RT, 3.0 µL of 0.1 mg/mL RGO solution was dropped on the surface of Au
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NPs/SPE, irradiated in the infrared light for 30 min. Afterwards, 2.5 µL of 10 mmol/L
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TH was added to the surface of RGO/Au NPs/SPE and dried at RT. Following that,
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3.0 µL of 5.0 µmol/L AFP-aptamer solution was dropped on the surface of
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TH/RGO/Au NPs/SPE and incubated for 2 h at RT, and then washed twice with PBS
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(0.1 mol/L, pH 7.0) to remove the excess aptamer. Finally, 0.5% BSA was used to
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block the possible remaining active sites against nonspecific adsorption and rinsed
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with PBS. Thus, the label-free electrochemical aptasensor (AFP-aptamer/TH/RGO/Au
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NPs/SPE) was fabricated, and stored in a refrigerator at 4 ºC before use.
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2.4. Electrochemical measurement the AFP concentration with the label-free
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electrochemical aptasensor
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To carry out the electrochemical measurement, 3.0 µL of different AFP
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concentration was dropped onto the surface of AFP-aptamer/ TH/RGO/Au NPs/SPE.
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After incubated for 60 min at 25 ºC, the electrode was washed twice with PBS. Then,
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differential pulse voltammetry (DPV) was performed in PBS (0.2 mol/L, pH 6.5)
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from -0.7 to -0.25 V to record the peak currents of the aptasensor for quantitative
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analysis. Each sample was detected for three times and the results were calculated as
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mean±RSD.
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3. Result and discussion
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3.1. Analytical principle of the label-free electrochemical aptasensor for AFP
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detection
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ACCEPTED MANUSCRIPT TH/RGO/Au NPs/SPE for AFP detection, and the analytical principle was illustrated in
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Fig. 1A. Firstly, a layer of Au NPs was deposited on the surface of the SPE by
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electrodeposition. Secondly, RGO was loaded on the surface of Au NPs/SPE due to the
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electrostatic adsorption. Following that, TH was assembled to the RGO/Au NPs/SPE
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through electrostatic interactions. Then, AFP-aptamer was immobilized on the surface
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of TH/RGO/Au NPs/SPE by electrostatic adsorption due to TH containing amino
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groups and easy to intercalate with nucleic acid molecules [35, 36]. When AFP
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solution was dropped onto the surface of AFP-aptamer/TH/RGO/Au NPs/SPE, the
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AFP-aptamer quickly recognized AFP via specific recognition reaction, which made
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the change of electrochemical signals of TH obtained by DPV. The value of
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electrochemical signal increased accordingly with the increase of the concentration of
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AFP. Thus, a high sensitivity of the label-free electrochemical aptasensor could be
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achieved.
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Herein, TH/RGO/Au NPs was employed as a nanocarrier to load AFP-aptamer.
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The decorated TH on the electrode acted as not only a bridging molecule to effectively
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capture and immobilize AFP-aptamer, but also the signal indicator for monitoring the
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concentration of the AFP. The electrochemical signal of TH could be amplified
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effectively by the synergistic effect between Au NPs with good electrocatalytic
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activity and RGO with high specific surface area.
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Fig. 1B illustrated the feasibility of the developed aptasensor for AFP detection
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using the DPV method. Seen from Fig. 1B, there was a remarkable current response
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(67.00 µA) at -344 mV in the presence of AFP (curve a). As a control, there appeared a
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mild current response (36.00 µA) at -348 mV without AFP (curve b). This may be due
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to the following reasons: The AFP-aptamer firstly covered the electrode surface which
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may hinder the electron transfer and the current was mild. When AFP solution was 8
ACCEPTED MANUSCRIPT dropped onto the surface of AFP-aptamer/TH/RGO/Au NPs/SPE, the AFP-aptamer
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was binding with AFP molecules via specific recognition reaction, and formed the
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aptamer-antigen complexes. The produced complexes were arranged with stable spatial
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structure on the surface of the electrode, providing more space for electron transfer,
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thus the current increased.
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3.2. Electrochemical characterization of the label-free electrochemical aptasensor
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In order to monitor the changes of electrochemical behavior at different stages of
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the aptasensor, CV was performed in PBS (pH 7.0, 0.1 mol/L Na2HPO4/KH2PO4 and
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0.1 mol/L NaCl) with scanning range from -1.0 to 1.0 V and scanning rate of 100 mV/s
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and the results were shown in Fig. 1C. The bare SPE (curve a) had no significant redox
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peaks current without electroactive material in the electrode. After the Au NPs was
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electro-deposited on the surface of SPE, an obvious cathodic peak appeared (curve b)
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because of the highly conductivity of Au NPs. Compared to curve b, the cathodic peak
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current was slightly decreased when the RGO was integrated on the surface of Au
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NPs/SPE (curve c), owing to the formation of a thin RGO film on the electrode.
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Subsequently, the reversible reduction and oxidation peaks with cathodic and anodic
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peak potentials of TH were observed (curve d), which indicating that TH could served
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as an electrochemical indicator for detection. After incubation with the AFP-aptamer,
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an obvious decrease of the peak current was observed (curve e), this may be the large
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resistance of the aptamer blocked the ability of electron transfer of TH at the sensing
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interface. The results also indicated that the electrostatic interaction between TH and
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Au NPs was very strong, and that AFP-aptamer could be firmly immobilized on the
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electrode surface. Finally, the cathodic and anodic peaks’ current obviously increased
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(curve
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when
AFP
solution
was
dropped
onto
the
surface
of 9
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AFP-aptamer/TH/RGO/Au NPs/SPE. Since the specific recognition between
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aptamer-antigen which provided the space for electron transfer and accelerated the
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electron transfer rate. What’s more, electrochemical impedance spectroscopy (EIS), a valid method for
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exploring the properties of the surface of the modified electrodes, was performed at
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0.24V in 10.0 mmol/L K3Fe(CN)6/K4Fe(CN)6 containing 0.1 mol/L KCl solutions at
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the frequency range from 1 to 100 KHz, and the results were shown in Fig. 2A. Seen
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from Fig. 2A, a large semicircle can be observed for the bare SPE (curve a), which
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implied that the impedance of the bare SPE was relatively high and displayed low Ret
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(electron transfer resistance). When Au NPs was deposited on the SPE, the Ret
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decreased obviously (curve b), implying that the Au NPs could greatly accelerate the
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electron transfer. After RGO and TH were modified on the surface of Au NPs/SPE, the
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semicircle further reduced (curve c and curve d), due to the upstanding electric
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conductivity of RGO and TH. Compared to the curve d, the Ret increased remarkably
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after the AFP-aptamer was immobilized (curve e), and decreased again when the
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specific recognition reaction between aptamer-antigen occurred (curve f). The EIS
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results were in good agreement with those obtained from CV, further revealing the
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successful fabrication of the label-free electrochemical aptasensor.
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3.3. Raman characterization of the label-free electrochemical aptasensor Fig. 2B depicted the Raman spectroscopy for the different stages of the electrode
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modification. As can be seen from Fig. 2B, there are two main characteristic Raman
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bands in 1350 cm-1 (D-band) and 1580 cm-1 (G-band), respectively. Compared to the
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curve b, the signal strength of curve c was significantly enhanced. By calculation, the
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value of ID/IG for RGO/Au NPs/SPE was 0.97, indicating that RGO successfully fixed 10
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on the electrode. After fixed TH and AFP-aptamer, the intensity of Raman bands did
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not change much, implying that RGO did not fell down from the surface of electrode.
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3.4. SEM characterization of the label-free electrochemical aptasensor Fig. 2C-2H demonstrated SEM images of the process of the label-free
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electrochemical aptasensor at different stages. In Fig. 2C, the bare SPE gave a uniform
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dark image without particles on the surface. There were homogeneously distributed
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spherical particles with the diameter of 80 ± 10 nm in the image of Au NPs/SPE (Fig.
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2D), indicating the successful deposition of Au NPs onto the surface of SPE. Fig. 2E
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depicted the SEM image of the RGO/Au NPs/SPE. The white spherical particles
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became smaller and blurred compared to the image of Au NPs/SPE, since the RGO
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was present on the surface of the Au NPs/SPE with a thin film. In addition, a substance
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similar to the shape of the branches could be clearly observed on the surface of
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TH/RGO/Au NPs/SPE (Fig. 2F), implying that TH binded with RGO and Au NPs
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firmly. When the AFP-aptamer was immobilized on the surface of TH/RGO/Au
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NPs/SPE, no apparent structure was seen in Fig. 2G. Fig.2H was the SEM image of the
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SPE after AFP added. Because of the smaller particle size of AFP molecules, the
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difference between Fig. 2G and Fig. 2H was not significant.
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3.5. XPS characterization of the label-free electrochemical aptasensor
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To further analyze the fabrication process of the aptasensor, X-ray photoelectron
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spectroscopy (XPS) measurement was performed. Four main features including O1s,
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N1s, C1s and Au4f peaks of the XPS spectrum were inspected (Fig. 3). Seen from Fig.
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3, Compared curve a with curve b, the Au4f (82.70 eV) signal increased sharply, while
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C1s (283.40 eV) signal decreased slightly due to the electrolytic deposition of Au NPs.
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After RGO was dropped onto the surface of Au NPs/SPE, the signal of C1s (283.20 eV)
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rised and Au4f (82.70 eV) signal decreased (curve c). However, the further decrease of 11
ACCEPTED MANUSCRIPT Au4f (82.20 eV) peaks accompanied with the increase in the peak intensities of N1s
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(397.70 eV) and O1s (530.70 eV) appeared with TH decoration (curve d),
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demonstrating that TH has been successfully attached to the surface of RGO/Au
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NPs/SPE. After immobilization of AFP-aptamer and AFP, the total intensity of O1s
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(531.00 eV) and N1s (398.00 eV) increased continuously, which promoted the decrease
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of C1s (284.00 eV) signal and the disappearance of Au4f (83.00 eV) signal (curve e and
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curve f). The XPS data fully confirmed that the biological molecules were correctly
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immobilized on the electrode surface.
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A further insight can be obtained from the inspection of table inserted in Fig. 3A,
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which summarized the relative abundance (% of atoms) of the various elements (O, N,
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C, Au). From a to f, a strong increase in the proportion of O from 16.56% to 38.49%
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and a bit increase in the proportion of N from 8.06% to 11.33% while a mild decrease
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in the proportion of C from 75.38% to 50.18%. In addition, the proportion of Au
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decreased strongly from 8.30% to 0.08%.
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3.6. Optimization of experimental conditions for detection AFP using the
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label-free electrochemical aptasensor
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To achieve the best results for the sensor, the experimental conditions, such as the
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incubation time, the pH of the buffer solution, incubation temperature and the
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concentration of AFP aptamer, were optimized. Fig. 4A showed the effect of the
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incubation time on the current response of the aptasensor. Seen from Fig. 4A, the
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current response value increased (from 56.69 µA to 61.65 µA) with the increase of the
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incubation time from 15 min to 30 min, and reached maximum (71.99 µA) when the
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incubation time was 60 min. When the incubation time exceeded to 60 min, the current
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response value tended to decrease (56.44 µA). Therefore, the incubation time was
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selected for 60 min for the further experiment. Fig. 4B depicted the influences of the incubation temperature on the current
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response of the aptasensor. It has been known that the higher and lower temperature
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were all harmful to the biomolecule activity. The response value of the biosensor
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increased from 43.97 µA to 72.00 µA with the incubation temperature increase from 4
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to 25 ºC. When the temperature was 25 ºC, the corresponding current reached the
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maximum (72.00 µA). As the temperature continued to rise, the response current
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decreased. Therefore, 25 ºC was selected for the optimal temperature.
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The pH of the buffer solution is a vital parameter for the aptasensor because the
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activity of the immobilized protein could be influenced by the acidity of the solution.
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So the aptasensor was tested in a series of PBS solution with the pH values varying
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from 5.7 to 8.0. As shown in Fig. 4C, with increasing pH value from 5.7 to 6.5, the
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current responses increased (from 50.87 µA to 72.54 µA), and then the value decreased
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(61.67 µA, 48.63 µA, 45.45 µA) rapidly at the higher pH values (7.0, 7.5, 8.0). As a
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consequence, the optimal pH 6.5 was used for further experiment.
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The effect of the concentration of AFP-aptamer on the current response of the
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aptasensor was shown in Fig. 4D. It can be clearly observed that the current response
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increased from 50.18 µA to 71.41 µA with increasing concentration of AFP-aptamer
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and reached a maximum (71.41 µA) at 5.0 µmol/L, implying that AFP-aptamer and
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AFP reacted completely. Due to the excess aptamer could hinder electron transfer,
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higher AFP-aptamer concentration made the response decrease. Therefore, 5.0 µmol/L
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was selected as the optimum concentration of AFP-aptamer.
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ACCEPTED MANUSCRIPT standard solutions using the DPV method. Under the optimized conditions, the
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developed aptasensor was used to detect different concentrations of AFP and the
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current response was recorded. Fig. 5A showed the electrochemical signal responses
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of the proposed aptasensor for the detection of AFP concentration in the range of
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0.1-100.0 µg/mL. Seen from Fig. 5A, the current response of the aptasensor increased
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along with the increasing concentration of AFP, which confirmed the formation
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between the AFP and the AFP-aptamer. As the concentration of AFP increasing, a
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larger amount of AFP was specifically recognized by AFP-aptamer, leading to the
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gradually increasing electrochemical signal response. As can be seen from Fig. 5B, the
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current response was linearly proportional with the AFP concentration in the range of
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0.1 - 100.0 µg/mL. The regression equation was Y = 0.3471X + 47.984 (Y was the peak
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current and X indicated the concentration of AFP) with a correlation coefficient of
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0.9987. Specifically, the limit of detection (LOD) can be calculated at a signal/noise of
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3 based on the standard deviation of the response (σ) and the slope (B) of the calibration
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curve [37, 38]. According to the formula: LOD = 3(σ/B), LOD was determined to be
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0.05 µg/mL (where σ was the standard deviation in blank sample, n=3). Compared to
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other reported AFP immunosensors [11, 14, 39, 40], the proposed aptasensor showed a
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high detection limit, which may be the following reason: The affinity between
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AFP-aptamer and AFP was not strong enough and the reaction was not complete; In
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addition, the aptamer-antigen immunocomplexs had spatial stereoscopic effect and
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hindered electron transfer to make decrease of current change.
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Although the proposed aptasensor showed a high detection limit and can not fully
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meet the clinical requirements at present, the simple and low-cost of aptamer provided
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a new promising platform for the design of the highly sensitive detection method in
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clinical immunoassays. 14
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3.8. Reproducibility, specificity and stability of the label-free electrochemical
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aptasensor To evaluate the reproducibility of the aptasensor, five freshly prepared modified
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electrodes were employed for the detection of AFP (100.0 µg/mL). All five electrodes
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exhibited similar current response (54.96 µA, 54.72 µA, 48.93 µA, 59.55 µA, 52.44 µA)
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and the inter-assay relative standard deviation (RSD) was 3.9%, suggesting that the
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proposed aptasensor had quite good reproducibility for AFP detection.
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Moreover, some biological molecules, such as BSA, HSA, IgG and IgE, were
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chosen as interferences to evaluate the specificity of the aptasensor. Fig. 5C showed the
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DPV current response of the aptasensor to all the above interferences (100.0 µg/mL)
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instead of the AFP under the optimal conditions. Seen from Fig. 5C, when the test
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sample was BSA, HSA, IgG and IgE, the aptasensor's current response value were
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40.02 µA, 38.38 µA, 33.22 µA and 32.94 µA, respectively, accounting for 48.19%,
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45.78%, 39.75%, 38.55% of the current response value of AFP (82.93 µA),
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respectively. The result showed that the aptasensor exhibits good specificity for AFP.
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Additionally, the stability of the biosensor was studied after storage at 4 ºC for 7
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and 14 days, respectively. The current response were 78.73 µA and 74.23 µA. The
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activity of the immunosensor can be maintained at 95.76% and 90.86%, demonstrating
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good stability of the aptasensor.
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3.9. Real sample analysis
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In order to verify the practical application of the fabricated sensor, the feasibility
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of the proposed method for the detection of human serum was evaluated by standard
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addition method. The human serum sample was donated by the local hospital, and the
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level of AFP in this sample was calculated to be 10.6 ng/mL by ELISA. 3 µL of known 15
ACCEPTED MANUSCRIPT quantified AFP solution (15.0 µg/mL, 20.0 µg/mL, 50.0 µg/mL) was dropped onto the
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surface of AFP-aptamer/TH/RGO/Au NPs/SPE. Then, 100.0 µL of human serum
3
sample were added to 5 mL of PBS solution (0.2 mol/L, pH 6.5), the levels of AFP in
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spiked samples were parallelly assayed for 3 times with DPV using the developed
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aptasensor. The experimental results were shown in Table 1. Results can be seen from
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Table 1 that the recoveries were in the range from 101.52% to 107.95%. It can be
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known that the recovery results were high which could meet the needs of the actual
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sample detection.
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9 Conclusions
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In summary, a label-free electrochemical aptasensor for the detection of AFP
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using TH/RGO/AuNPs as the immobilization platform and AFP aptamer as the
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recognition molecule has been successfully developed. Combining Au NPs (high
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conductivity and biocompatibility), RGO (large specific surface area and upstanding
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electric conductivity), TH (electrochemical indicator) and AFP aptamer (high affinity
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and specificity), the proposed aptasensor showed excellent performance with highly
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selective, simple operation and long-term stability. In the range of 0.1 to 100.0 µg/mL,
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a linear curve was obtained (Y=0.3471X + 47.984) between current and AFP
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concentrations with a readily achievable detection limit of 0.050 µg/mL at a
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signal/noise ratio of 3. Although the proposed aptasensor showed a high detection limit
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and can not fully meet the clinical requirements at present. It was worth pondering that
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we need to further optimize the sensor preparation process to improve the detection line.
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Most importantly, the simple and cost-effective sensing strategy provides a new
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promising platform for the design of the highly sensitive detection method, showing
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potential application for aptamer in clinical immunoassays.
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Acknowledgments This work was supported by the National Nature Science Foundation of China
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(Nos. 81460451, 81760534 and 81430055), the Innovation Project of GUET Graduate
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Education (2017YJCX95, YCSW2017146), the Appropriate Health Technology
5
Development Project of Guangxi Zhuang Autonomous Region (No. S201422-03), the
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National
7
2015GXNSFDA139025, 2016GXNSFAA380011 and 2016GXNSFAA380080) and
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the Foundation of Guangxi Key Laboratory of Automatic Detecting Technology and
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Instruments (No. YQ17114).
of
Guangxi
province
of
China
(Nos.
SC
Foundation
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ACCEPTED MANUSCRIPT Figure captions
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Fig. 1 (A) Principle of the label-free electrochemical aptasensor for detection AFP
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based on AFP-aptamer and TH/RGO/Au NPs. (B) DPV response of the label-free
4
electrochemical aptasensor with AFP (a) and without AFP (b). The concentration of
5
AFP was 100.0 µg/mL and the DPV signal was obtained in PBS (0.2 mol/L, pH 6.5)
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from -0.7 to -0.25 V. (C) CV response of (a) bare SPE, (b) Au NPs/SPE, (c) RGO/Au
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NPs/SPE, (d) TH/RGO/Au NPs/SPE, (e) AFP-aptamer/TH/RGO/Au NPs/SPE, (f)
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AFP/AFP-aptamer/TH/RGO/Au NPs/SPE.
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Fig. 2 (A) Electrochemical impedance spectroscopy of (a) bare SPE, (b) Au NPs/SPE,
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(c) RGO/Au NPs/SPE, (d) TH/RGO/Au NPs/SPE, (e) AFP-aptamer/TH/RGO/Au
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NPs/SPE, (f) AFP/AFP-aptamer/TH/RGO/Au NPs/SPE. (B) Raman spectra of (a) bare
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SPE, (b) Au NPs/SPE, (c) RGO/Au NPs/SPE, (d) TH/RGO/Au NPs/SPE, (e)
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AFP-aptamer/TH/RGO/Au NPs/SPE, (f) AFP/AFP-aptamer/TH/RGO/Au NPs/SPE.
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(C) SEM image of bare SPE. (D) SEM image of Au NPs/SPE. (E) SEM image of
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RGO/Au NPs/SPE. (F) SEM image of TH/RGO/Au NPs/SPE. (G) SEM image of
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AFP-aptamer/TH/RGO/Au
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AFP/AFP-aptamer/TH/RGO/Au NPs/SPE.
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Fig. 3 (A) Full XPS spectra of the surface of SPE in different stages. (B) XPS spectra of
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C1s, O1s, N1s and Au4f. Among them (a) bare SPE, (b) Au NPs/SPE, (c) RGO/Au
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NPs/SPE, (d) TH/RGO/Au NPs/SPE, (e) AFP-aptamer/TH/RGO/Au NPs/SPE, (f)
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AFP/AFP-aptamer/TH/RGO/Au NPs/SPE.
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Fig. 4 (A) Effect of incubation time on the current signals. (B) Effect of incubation
23
temperature on the current signals. (C) Effect of pH on the current signals. (D) Effect of
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the concentration of aptamer on the current signals. The concentration of AFP was
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100.0 µg/mL and the DPV current signal was obtained in PBS solution (0.2 mol/L, pH
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(H)
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ACCEPTED MANUSCRIPT 6.5) from -0.7 to -0.25 V with a 100 mV/s scanning rate. And the error bars represent
2
the relative standard deviation (RSD) (n=3 electrodes).
3
Fig. 5 (A) Electrochemical signal responses of the label-free electrochemical
4
aptasensor for the detection of different concentrations of AFP including 100.0, 80.0,
5
65.0, 50.0, 40.0, 25.0, 10.0, 1.0 and 0.1µg/mL. (B) Calibration curve of the aptasensor
6
for the detection of different concentrations of AFP. (C) Specificity of the proposed
7
aptasensor with BSA, HSA, IgG and IgE instead of AFP. The current signal was
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obtained by DPV which was carried out in PBS (0.2 mol/L, pH 6.5) from -0.7 to -0.25
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V with a 100 mV/s scanning rate. And the error bars represented the relative standard
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Tables
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Table 1 Determination of AFP by the proposed aptasensor in PBS (0.2 mol/L, pH 6.5)
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containing healthy human serum from -0.7 to -0.25 V with a 100 mV/s scanning rate
AFP Added
Averange
(ng/mL)
(µg/mL)
(µg/mL)
15.00
16.12
20.00
21.59
50.00
50.76
Recovery
(%)
(%)
1.13
107.47%
0.36
107.95%
3.47
101.52%
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AFP in samples
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ACCEPTED MANUSCRIPT Hightlights A simple label-free electrochemical aptasensor based on TH/RGO/Au NPs was developed.
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TH/RGO/Au NPs was the immobilization platform for capture the AFP-aptamer.
The electrochemical responses were proportional to the AFP concentration.
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The proposed aptasensor showed facile procedure, low cost and high
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selectivity.