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Label-free electrochemical detection of HepG2 tumor cells with a self-assembled DNA nanostructure-based aptasensor
Accepted Manuscript Title: Label-free electrochemical detection of HepG2 tumor cells with a self-assembled DNA nanostructure-based aptasensor Authors: Duanping Sun, Jing Lu, Dabin Chen, Yunfei Jiang, Zhiru Wang, Weiwei Qin, Yanyan Yu, Zuanguang Chen, Yuanqing Zhang PII: DOI: Reference:
S0925-4005(18)30846-3 https://doi.org/10.1016/j.snb.2018.04.142 SNB 24618
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
9-1-2018 20-4-2018 24-4-2018
Please cite this article as: Duanping Sun, Jing Lu, Dabin Chen, Yunfei Jiang, Zhiru Wang, Weiwei Qin, Yanyan Yu, Zuanguang Chen, Yuanqing Zhang, Label-free electrochemical detection of HepG2 tumor cells with a self-assembled DNA nanostructure-based aptasensor, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.04.142 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.
Label-free electrochemical detection of HepG2 tumor cells with a self-assembled DNA nanostructure-based aptasensor
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Duanping Suna,1, Jing Lua,1, Dabin Chena,1, Yunfei Jianga, Zhiru Wanga,
a
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Weiwei Qina, Yanyan Yub, Zuanguang Chena,*, Yuanqing Zhanga,*
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, Guangdong
School of Pharmacy, Nantong University, Nantong, Jiangsu 226001, China
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* Corresponding authors.
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b
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510006, China
1
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(Y. Zhang).
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E-mail addresses: [email protected] (Z. Chen), [email protected]
These authors contributed equally to this work.
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Graphical Abstract
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Highlights
Aptasensor was developed for the electrochemical detection and release of CTCs. Tetrahedron-based aptamer was used for the highly enhanced capture of
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target cells.
Dendritic structure nanoprobes amplified the electrochemical signals
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significantly.
The detection limit of the cytosensor could be estimated as 5 cells mL−1 for
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This method released the captured HepG2 cells with little damage for further
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HepG2.
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study.
Abstract
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The development of rapid, sensitive and convenient methods for the monitoring of rare circulating tumor cells (CTCs) is of great significance in cancer diagnostics and therapy.
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Herein a label-free and competitive electrochemical aptasensor was proposed for the efficient capture, ultrasensitive detection and controlled release of CTCs. Firstly, the DNA nanotetrahedron (NTH)-based TLS11a aptamer probe was immobilized on a disposable screen-printed gold electrode (SPGE) surface via the Au−S bonds for the 2
highly enhanced capture of liver cancer HepG2 cells. Then, hybrid nanoprobes of PdPt nanocages labeled with complementary DNA (cDNA), hemin/G-quadruplex DNAzyme and horseradish peroxidase (HRP) were attached on the SPGE substrate by the DNA hybridization, resulting in the formation of dendritic structure (DS)
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nanoprobes with self-assembly methods for the greatly enhanced sensitivity. When the target HepG2 cells existed, they can compete with DS nanoprobes to bind with NTH-
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based aptamer probe, resulting in the release of the DS nanoprobes from the SPGE. This method exhibits ultrahigh selectivity and sensitivity toward HepG2 with detection
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limits of 5 cells per ml. Furthermore, our strategy allows for easy detachment of the
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captured cells from the SPGE without compromising cell viability by an
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electrochemical cleavage of the Au–S bonds. The present study provides a label-free
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technique for highly selective and efficient quantification of tumor cells, which is
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essential in the early cancer diagnosis and treatment of cancer. Keywords: electrochemical cytosensor; aptamer, HepG2 cells; nanotetrahedron;
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dendritic structure nanoprobes
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1. Introduction
Cancer is a highly fatal disease and has become one of the biggest killers of human
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health in the world [1-3]. Circulating tumor cells (CTCs), a type of cancer cells that can circulate in the peripheral blood or through the lymphatic system, can lead to a new tumor metastasis. Numerous clinical studies has indicated that CTCs can act as a biomarker in the early detection of cancer, cancer diagnosis and therapy assessment 3
[4,5]. However, the number of tumor cells circulating in human blood are exceedingly rare (1–3000 CTCs per mL against 109 red blood cells per mL) [6]. Thus there is an urgent demand to capture and detect these rare tumor cells accurately. Up to now, various strategies have been designed to determine the cancer cells,
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including fluorescent measurements or imaging [7], flow cytometry [8], magnetic immunoassay [9], electrochemiluminescence [10,11], and electrochemistry [12-17].
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Among them, electrochemical biosensor, especially aptamer-based biosensor
(aptasensor), has received particular attention in cancer cell research due to the intrinsic
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advantages in terms of excellent portability, rapid response and sensitive recognition
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[18-23]. Aptamers are designed through systematic evolution of ligands by exponential
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enrichment (SELEX) system in vitro and single-stranded nucleic acids with specific
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three dimensional structure. They have high specificity and affinity toward the target
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molecules ranging from small molecules like proteins to whole cells [24]. However, because of the flexibility of single-stranded nucleic acids, it tends to undergo self-
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assembled monolayer aggregation by immobilizing aptamers on substrates, which largely impede accessibility for the target tumor cells. It is also difficult to control the
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spatial orientation of single-stranded nucleic acids with precision [21]. As a result, the analytical selectivity and sensitivity is not high enough for cancer cells and it fail to
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preserve accuracy when challenged with a clinical sample. Therefore it is highly desirable to develop a reliable and rapid platform to analysis tumor cells specifically and sensitively with modest requirements of sample volumes. To enhance the selectivity and sensitivity of electrochemical aptasensor, various 4
methods have been employed to modify working electrode [25-27]. DNA nanotechnology is emerging as a novel nanotechnology with several unique advantages including a highly programmable nature, ease of preparation and high precision. On the basis of these, DNA nanotetrahedron (NTH) is a classical three-dimensional framework,
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which was originally reported by Turberfield and co-workers [28]. Due to the powerful structural rigidity and excellent biocompatibility, DNA NTH can be an ideal strategy
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for the electrochemical aptasensing platform [29, 30]. For example, Fan and colleagues
developed a series of electrochemical biosensors based on self-assembled DNA NTH
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nanostructures for greatly increasing target accessibility and improving the sensitivity.
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As a consequence of the attractive features of NTH, DNA tetrahedron-based aptamer
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probe can provide a rigid scaffold for cancer cell recognition with high selectivity and
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high reproducibility [31,32]. However, only by using DNA NTH-based aptamer for
clinical samples.
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electrochemical cytosensing [21], the detection sensitivity is not good enough for the
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In addition, through the DNA hybridization with layer-by-layer (LBL) assembly process, dendritic structure (DS) DNA is an excellent strategy for immobilizing
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abundant signal tags, nanomaterials or biomolecules, such as nature enzymes (peroxidase and glucose oxidase) and mimic enzymes (hemin/G-quadruplex DNAzyme)
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[33]. Meanwhile, because of the excellent electrocatalytic capacity through in situ forming the DS biomolecules or nanomaterials, DSDNA provides a very favorable method for electrochemical signal amplification [34]. As a consequence of the excellent stability, DSDNA has garnered particular attention in designing electrochemical 5
aptasensors [35]. Due to the DSDNA with enzymatic amplification, we can successfully achieve the efficient improvement of detection sensitivity of the electrochemical aptasensing platforms for tumor cells. Screen-printing is one of the most important methods towards rapid, simple and inexpensive production of electrochemical
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biosensors. Screen-printed electrodes with speed of mass production and low cost have often been used for developing electrochemical biosensors and improving their
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performances [36]. Their main advantage is the reduction of sample volume required,
which in turn helps in reducing the overall size of the biosensing platform into which
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the device will be integrated. Furthermore, SPEs can avoid some of the common
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questions of classical solid electrodes, such as tedious cleaning processes and memory
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effects.
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Herein, we for the first time combined the strengths of advanced aptamer strategy,
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DNA-based nanotechnology and disposable screen-printed electrodes to develop a label-free and competitive electrochemical aptasensor for the capture, detection and
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release of HepG2 tumor cells (Scheme 1). Firstly, we incorporated an expanded nucleotide-containing aptamer into a DNA NTH structure for greatly enhanced capture
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of cancer cells. Then the self-assembled NTH-based TLS11a aptamer probe [13,22] was immobilized on a disposable screen-printed gold electrode (SPGE) surface via
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Au−S interactions. Nanoprobes (nanoprobe1 and nanoprobe2) were fabricated by PdPt nanocages labeled with two complementary DNA (cDNA1 and cDNA2), horseradish peroxidase (HRP), and hemin/G-quadruplex DNAzyme (Scheme 1A). The nanoprobes were captured onto the SPGE surface via the DNA hybridization, resulting in the 6
formation of DS nanoprobes for the greatly enhanced sensitivity. When the target cells existed, NTH-based TLS11a aptamer probe efficiently captured the target HepG2 cells, resulting in the denaturation of double-stranded DNA and the release of the DS nanoprobes. Finally, captured HepG2 cells can be released from the SPGE for further
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analysis by an electrochemical cleavage of the Au–S bonds via a negative potential (Scheme 1B). In this way, the proposed electrochemical aptasensor showed excellent
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selectivity and sensitivity, indicating potential applications in the early diagnosis and treatment of cancers and choice of individual sensitive anti-cancer drugs.
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Scheme 1
(HPLC
purified)
were
synthesized
from Sangon
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All oligonucleotides
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2.1. Materials and apparatus
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2. Experimental section
Biotechnology Co., Ltd. (Shanghai, China) with/without thiol labeling. The sequences
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of the oligonucleotides were listed in Table S1 (Supplementary material). Other detailed chemicals or instruments are provided in
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the Supplemental material. All reagents were of analytical grade and were used without further purification. All solutions were prepared with ultrapure water (18.2 MΩ∙cm,
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Milli-Q, Millipore). The electrochemical detection was performed on disposable screen-printed gold electrode (SPGE) with 4 mm diameter working area (DropSens220AT, LLanera, Spain). 2.2. Blood sample preparation 7
Different number of HepG2 cells were spiked into the blood, which was collected from healthy volunteers. The whole blood samples were centrifuged at 1500 rpm for 5 min, then the supernatant was discarded. After this step, 1 ml of red blood cell lysis buffer were added into blood, and incubated at room temperature for 5 min. Finally, the
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sediment was washed and resuspended in PBS solution to obtain a cell suspension. 2.3. Preparation of nanoprobes
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According to the reported method [35,37], Pd-Pt nanocages were prepared.
Detailed steps are provided in the Supplemental materials. 50 μL cDNA1 (5 μM) or
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cDNA2 (5 μM) added in 1 mL Pd-Pt nanocages was stirred for 12 h at 4 °C.
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Subsequently, 50 μL of HRP (1 mg mL-1) were added to the solution and stirred for 6 h
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to form nanoprobes of cDNA1-HRP-Pd-Pt nanocages or cDNA2-HRP-Pd-Pt
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nanocages. Then hemin (0.1 mg) was added to the solution (pH 7.0) for 2 h at 4 °C to
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form bioconjugates of hemin/G-quadruplex DNAzyme. After the supernatant was discarded, the nanoprobe of hemin/G-quadruplex-cDNA1-HRP-Pd-Pt nanocages
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(nanoprobe1) or hemin/G-quadruplex-cDNA2-HRP-Pd-Pt nanocages (nanoprobe2) was obtained and redispersed in 500 mL PBS (10 mM, pH 7.4) solution at 4 °C for
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further use.
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2.4. Preparation of nanotetrahedra The nanotetrahedra were assembled from the three thiolated DNA strands of 61
nucleotides (THA, THB, and THC) and one aptamer-containing DNA strand of 125 nucleotides (THD). Before the assembly of nanotetrahedra, the monomers (THA, THB, THC, and THD) were pretreated with 10 mM TCEP for 2 h at room temperature (RT) 8
to cleave the disulfide linkage. Then the DNAs were mixed equivalently in TM buffer, heated at 95 °C for 10 min, and rapidly cooled at 4 °C for 2 min. The formation of DNA nanostructures was analyzed using 3% agarose gel electrophoresis in TBE buffer at 100 V for 25 min.
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2.5. Fabrication of electrochemical aptasensor The fabrication process of the proposed aptasensor for HepG2 cells was shown in
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Scheme 1B. 10 μL of 500 nM NTH-based TLS11a aptamer probe was immobilized on
the surface of SPGE at RT for 2 h, and the unbound aptamer probes were thoroughly
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washed away with ultrapure water. After immobilization, the blank sites on the SPGE
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were then blocked with 1 mM 6-mercapto-1-hexanol (MCH) for 2 h. Unbound MCH
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was also washed away with ultrapure water. Finally, 10 μL nanoprobe1 and 10 μL
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nanoprobe2 were dropped onto the obtained SPGE surface for 2 h via the hybridization
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of aptamer and cDNA1, resulting in the formation of DS nanoprobes via the hybridization of cDNA1 and cDNA2. Each step was characterized by differential pulse
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voltammetry (DPV). As a control, only 10 μL nanoprobe1 were dropped on the SPGE surface and incubated under the same conditions.
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2.6. Cell capture and electrochemical detection A 200 μL HepG2 cells with a certain concentration in PBS solution was dropped
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on the modified SPGE and incubated at 37 °C for 1 h to capture the target cells. Then the SPGE was carefully washed with 10 mM PBS for three times. To determine the cell viability, the captured cells on the SPGE were stained with calcein acetoxymethyl ester (calcein-AM, a cell viability indicator). As a control, a 200 μL of PBS buffer containing 9
MCF-7 cells and a 200 μL 1:1 mixture of MCF-7 cells and HepG2 cells were dropped on the SPGE surface and incubated under the same conditions. For electrochemical detection, the SPGE was placed in 3 mL of 100 mM PBS (pH 7.0) solution containing 3 mM hydroquinone (HQ) and 2.0 mM H2O2. DPV analysis was performed from 0.2 to
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−0.3 V at 50 mV pulse amplitude. 2.7. Cell release and viability analysis
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After electrochemical detection, a negative potential was performed to break Au–
S bonds on the SPGE to release the captured HepG2 cells. The cyclic voltammetry (CV)
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was carried out in the PBS (10 mM, pH 7.4) solution, ranging from -0.9 to -1.7 V at a
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scan rate of 50 mV s-1. Released cells were observed and counted using a microscope
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system. Furthermore, cell viability were determined by trypan blue staining assay. After
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the cells were released, the remaining cells solution were recollected by centrifugation
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for 5 min at 1000 rpm, and redispersed in PBS (10 mM, pH 7.4) for staining assay experiment. The released cells were immersed into a trypan solution (0.04%) for 3 min.
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By trypan blue staining, viable cancer cells and dead cancer cells can be observed using
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a microscope system.
3. Results and discussion
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3.1. Principle of the cytosensor for HepG2 detection The self-assembled DNA nanostructure-based electrochemical aptasensor for capturing, detecting and releasing of tumor cells was illustrated in Scheme 1. In this work, Pd-Pt nanocages, with HRP-mimicking catalytic activity and high surface area, 10
were used as the nanocarriers for loading the amount of biomolecules [35, 37]. The cDNA consisted of two sequences (DNA hybridization sequences and G-quadruplexforming sequences, as shown in Table S1). In the presence of hemin, the catalytic Gquadruplex/hemin DNAzymes could be formed. G-quadruplex/hemin DNAzymes, a
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complex between hemin and a single-stranded guanine-rich nucleic acid, has been widely used as HRP-mimicking DNAzymes electrocatalysts for electrochemical
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cytosensing [12-14]. The nanoprobe involves the immobilization of complementary DNA (cDNA), hemin/G-quadruplex DNAzyme and HRP on the surface of Pd-Pt
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nanocages through a LBL assembly process (Scheme 1A). The signal amplification
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strategy of the electrochemical cytosensor was depended on the electrochemical feature
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of benzoquinone (BQ) produced from the oxidation of HQ with H2O2, which was
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catalyzed by Pd-Pt nanocages, HRP [38, 39] and HRP-mimicking G-quadruplex/hemin
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DNAzyme together. The process is shown as below H2O2 + H2Q → Q + 2H2O
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Q + 2H+ + 2e- → H2Q
where H2Q is hydroquinone and Q is benzoquinone.
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Then, we incorporated an expanded nucleotide-containing TLS11a aptamer into a DNA NTH structure to modify working electrode of SPGE for enhanced
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electrochemical analysis of CTCs. Nanoprobe1 can be attached on the SPGE through the hybridization of TLS11a aptamer and cDNA1. Meanwhile, nanoprobe1 and nanoprobe2 can form the DS nanoprobes by LBL assembly with the hybridization of cDNA1 and cDNA2. A large amount of HRP, HRP-mimicking DNAzyme and 11
nanocages of the formed DS nanoprobes can significantly amplify the electrochemical signal and improve the sensitivity. When the target cancer cells existed, NTH-based TLS11a aptamer probe can recognize and capture the target HepG2 cells. And it can result in the denaturation of double-stranded DNA structure and the release of the DS
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nanoprobes from the surface of SPGE. Finally, by using the electrochemical method (negative potential), the captured cancer cells were released from SPGE surface by
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cleaving the Au-S bonds on the SPGE surface, resulting in the further analysis of the
tumor cells (Scheme 1B). As a result, the selectivity and sensitivity of the
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electrochemical aptasensor is greatly enhanced based on the DNA NTH structure,
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3.2. Characterization of Pd-Pt nanocages
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aptamer method and the DSDNA strategy.
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The morphology of the prepared Pd-Pt alloy nanocages was characterized using
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scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. As can be seen from Fig. 1A, it is clear that the formed Pd-Pt nanocages showed
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cubic shape and coarse surface. And we also observed a darker contrast in the center, indicating the nanocages with porous walls and hollow interiors, as shown in Fig. 1B.
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From Fig. 1C and D, the formed Pd-Pt nanocages exhibited a slightly larger size than that of the Pd nanocubes. The diameters of the nanocages were uniformly distributed at
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approximately 32 nm (Fig. 1D) and average edge length of Pd nanocubes was about 20 nm (Fig. 1C). These results demonstrated the successful synthesis of Pd-Pt nanocages, and the morphology was acceptable compared to those reported [35, 37]. Figure 1 12
3.3. Characterization of aptamer and DNA nanotetrahedron (NTH) structure The TLS11a aptamer (Fig. S1) was selected for HepG2 cells, which can specifically bind to the protein expressed on the surface of human HepG2 cells [12,22].
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To allow a more direct visualization of TLS11a aptamer and HepG2 cells, fluorescence microscopy was performed to investigate HepG2 cells incubated with fluorescein
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isothiocyanate (FITC)-labeled TLS11a aptamer. As can be seen from Fig. S2, FITCTLS11a aptamer labeled HepG2 cells showed green fluorescence, providing solid
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evidence of the good binding ability of aptamer TLS11a to HepG2 tumor cells.
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As shown in Fig. 2A, NTHs, self-assembled from four single-stranded sequences,
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were introduced to enhance detection performance in this work. Briefly, TLS11a
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aptamers are built into one of the NTH sequences (THD), and the other three (THA,
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THB and THC) end with thiol groups were attached to the SPGE surface via Au−S interactions. Then the hybridization of with aptamer-based NTH and cDNA1 would
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result in the formation of new NTH structure. As shown in Fig. 2B, agarose gel electrophoretic method was performed to
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demonstrate our NTH structure. The NTH (lane 4) migrated more slowly than combinations constructed of fewer than four single-stranded sequences (lane 1, lane 2
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and lane 3). And the only major clear band can be observed on the gel, because of the rapid and specific DNA hybridization ensuring the high yield of NTHs. By observing lane 5 (the DNA hybridization of aptamer-based NTHs and cDNA1), a new and slower band, compared with that of the NTHs (lane 4), could be seen. In order to further 13
investigate the behavior of TLS11a, cDNA1 and cDNA2, agarose gel characterization was also performed. With the addition of strands from lane 1 to lane 3, it is clear that a significant reduction of electrophoretic mobility could be observed, which can be attributed to the increased molecular mass (Fig. S3). Therefore, the results
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demonstrated that our aptamer-based NTHs had been successfully formed and its response to the HepG2 tumor cells was further studied in the following electrochemical
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experiments.
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3.4. Electrochemical characterization of cytosensor
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Figure 2
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To understand the events occurring on the SPGE surface, CV and EIS
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characterization were performed. Fig. S4A and B shows the CV and EIS curves of the
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modified SPGE in the assembly processes. For the bare SPGE, a couple of reversible redox peaks were observed (curve a, Fig. S4A) and the Nyquist plot included no
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semicircle domain (curve a, Fig. S4B). However, after the modification of NTH-based aptamer, the gap between the cathodic and anodic peaks became wider and the peak
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current decreased obviously (curve b, Fig. S4A); meanwhile, a small semicircle was observed (curve b, Fig. S4B), suggesting the nonconductive DNA blocked the electron
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transfer. With further the modification of MCH, the peak current continuously decreased (curve c, Fig. S4A) and the semicircle gradually increased (curve c, Fig. S4B). After incubating with the nanoprobes, a larger decrease in the peak currents and a larger increase of the semicircle diameter could be seen (curve d, Fig. S4A and S4B), 14
indicating the bad conductivity of the nanoprobes. Finally, the aptasensor was incubated with the HepG2 cells, and the large amount of cancer cells still contributed to a decrease in the peak currents and a much larger semicircle (curve e, Fig. S4A and S4B). These
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results demonstrated the successful assembly of the electrochemical aptasensor.
3.5. Electrochemical characterization of nanoprobes
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Additionally, DPV characterization was utilized to demonstrate the signal amplification effect of the designed nanoprobes. As depicted in Fig. 3A, when the
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nanoprobe1/MCH/aptamer-NTH/SPGE was immersed in the 100 mM pH 7.0 PBS,
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electrochemical responses cannot be observed in the solution (curve a). When the
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nanoprobe1/MCH/aptamer-NTH/SPGE was placed in the 100 mM PBS solution with
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only 3 mM HQ, a weak DPV reduction peak appeared, which was ascribed to the
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electrochemical reduction of HQ (curve b). As can been seen in curve c, a large shift in the DPV peak was observed, when the nanoprobe1/MCH/aptamer-NTH/SPGE was
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placed in the 100 mM PBS solution with 2 mM H2O2 and 3 mM HQ. The results can indicate that the nanoprobes exhibits its good peroxidase catalytic activity by efficiently
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catalyzing the oxidation of HQ with H2O2. As can be seen from Fig. 3B, compared with the peak current of cDNA1-HRP-Pd-
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Pt nanocages (curve a), the nanoprobe1 with the HRP-mimicking DNAzyme, HRP and Pd-Pt nanocages together catalyzed oxidation of the redox-tags (HQ) with H2O2 and amplified the electrochemical signals (curve b, nanoprobe1). However, larger increase of DPV peak current was observed through the formation of the DS nanoprobes (curve 15
c, nanoprobe1 + nanoprobe2). Due to the A-T and G-C base pairs, a large amount of HRP-mimicking DNAzyme, HRP and Pd-Pt nanocages can be embedded into DS nanoprobes. Therefore, the DS nanoprobes effectively amplified the electrochemical signal due to the synergetic catalysis of numerous Pd-Pt nanocages, HRP and HRP-
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mimicking DNAzyme to the redox-tags (HQ) with H2O2.
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Figure 3
3.6. Optimization of the experimental conditions
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To achieve the optimum cell capture time, HepG2 cell solution was dropped onto
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the SPGE surface and incubated for different times (30, 45, 60, 75, 90 min). As shown
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in Fig. S5A, the current signal strengthened along increasing the cell incubation time.
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However, the minimum signal was achieved at 60 min; thus, 60 min was chosen as the
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optimal cell incubation time. We also investigated the concentration of HQ on the analytical performance of the electrochemical aptasensor. Fig. S5B displays the effect
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of the HQ concentration on the DPV responses. The signal gradually increased with increasing HQ concentration from 1.0 to 4.0 mM. With more than 3 mM, the current
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signal reached a plateau. Thus, the optimal HQ concentration was 3 mM in subsequent
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experiments.
3.7. Cell capture and electrochemical detection The high sensitivity plays an important role in the early clinical diagnosis. Under the optimum conditions, the current signal decreased along with the increasing 16
concentration of HepG2 cells in PBS buffer. Fig. 4A displays the DPV current signals after the detection of different concentration of HepG2 cells from 10 to 1×106 cells mL−1. There is a good linear correlation between the logarithm values of HepG2 cell concentration (Ccell) and the DPV current value. The linear equation is I (μA) =37.6896
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– 5.6055 lgCcell (cells mL−1) (R2 = 0.9961), as depicted in Fig. 4B. And the detection limit is calculated as 5 cells mL−1 for HepG2 cells by using 3σ method. The
electrochemiluminescence
immunosensor,
inductively
coupled
plasma
mass
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spectrometry and microcantilever aptasensor (Table 1).
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performance is better than that of previous methods for HepG2 cells, such as
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The high selectivity is another important criterion in the early cancer diagnosis. To
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further investigate the specificity of the aptasensor, breast cancer MCF-7 cells, liver
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cancer HepG2 cells and the mixture of HepG2 cells and MCF-7 cells were tested. As
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shown in Fig. 4C and D, compared with the blank control, no obvious current signal was observed for MCF-7 cells (105 cell mL-1). Moreover, when the aptasensor was performed to detect HepG2 cells and the mixture, there was conspicuous current signal
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for HepG2 cells (105 cell mL-1). The above results demonstrated that the selectivity of
Figure 4
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the aptasensor was satisfactory for the determination of target HepG2 cells.
Nest, to understand the repeatability of the aptasensor, relative standard deviations (RSD) were utilized to estimate the repeatability. According to the data from five independently fabricated SPGEs for the same cell concentration (104 cells ml-1), the 17
electrochemical cytosensor performed a RSD of 6.53%, demonstrating the acceptable repeatability of the cytosensor for the analysis of target HepG2 cells. In addition, the stability of the aptasensor was also studied. After storage at 4°C for one week, no obvious change in electrochemical signal was observed, which suggested a satisfactory
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stability of the nanostructured-based cytosensor. Furthermore, to explore the potential clinical application of the aptasensor, we
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spiked blood with various concentrations of HepG2 cells (103 and 105 cell mL−1). As shown in Table S2, the results displays the recovery of 87.3% and 85.1% and the RSD
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of 5.28% and 6.35%, respectively, indicating the good performance of the strategy.
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These results demonstrate that the electrochemical method provides a potential clinic
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diagnostic tool for the analysis of tumor cells in blood sample.
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3.8. Cell release and viability analysis
The successful release of CTCs from substrate without damage plays key role in
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subsequent study of cell analysis [44-46]. In this work, we uses an electrochemical desorption method for the efficient release of HepG2 tumor cells by cleaving the Au-S
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bonds on the SPGE interfaces. Before cell release, there were a large population of alive HepG2 cells on the substrate by observing the fluorescence signals (Fig. 5A). Fig. S6A
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and B shows the electrochemical curves of the original SPGE and regenerated SPGE by applying an electrical potential. Compared with the original SPGE, the regenerated SPGE had the same peak current and impedance spectra. Fig. S6C shows the black fluorescence image of the SPGE surface after cell release. There results suggested a 18
nearly 100% of the captured cells were released by this electrochemical method. To further investigate the cell viability of released cells (the ratio of alive cells to total cells), we treated the SPGE with the different electrochemical potential with -0.9, -1.1, -1.3, -1.5, and -1.7 V for 30s, respectively. The results in Fig. 5B suggested the
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viability decreased to 94% due to the little amount of dead cells. The released HepG2 cells from SPGE by the electrochemical method were further collected, and the
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microscope image was shown in Fig. S6D. The results demonstrates that the released cells still maintain high viability for further study.
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Figure 5
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4. Conclusions
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In conclusion, a label-free, portable and competitive electrochemical cytosensor
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was proposed for the capture, detection and release of HepG2 tumor cells. Compared with the previous methods with DNA tetrahedron-based aptamer [21, 31], we combined
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the strengths of DNA-based nanotechnology and DS nanoprobes (with a relatively large amount of enzyme) for highly efficient capture, ultrasensitive electrochemical detection
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and controlled release of the HepG2 cells in the SPGE. Due to the unique property of the DNA NTH-based aptamer probe and in situ LBL assembly of DS nanoprobes, the
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present method significantly improves selective and sensitive electrochemical performance for analyzing tumor cells. Moreover, the captured cells can be released with little damage by an electrochemical desorption method. The results show the high selectivity, acceptable sensitivity and good reliability. The low detection limit is 5 cells 19
per mL. This proposed strategy provides a powerful tool for comprehensive studies of CTCs and to show the great potential in early cancer diagnosis.
Acknowledgments
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This work was supported by the National Natural Science Foundation of China (No. 81601571 and 21675177), the Science and Technology Planning Project of Guangdong
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Province (No. 2016B030303002) and the Medical Scientific Research Foundation of
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Guangdong Province (No. A2017033).
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found in the online version.
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electrochemical release of circulating tumor cells by using aptamers modified gold nanowire
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Biographies Duanping Sun
received his Ph.D. degree in pharmaceutical analysis from Sun Yat-Sen University in 2016 under the
supervision of Professor Zuanguang Chen. He is presently employed as a senior research associate in School of Pharmaceutical
Sciences, Sun Yat-Sen University. His current research interests focus on electrochemical biosensor, microfluidic chip, and
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pharmaceutical analysis.
Jing Lu received her Ph.D. degree from Sun Yat-Sen University in 2017. She is presently employed as a senior research associate
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in School of Pharmaceutical Sciences, Sun Yat-Sen University. Her current research interests include electrochemical biosensor
and pharmacology.
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Dabin Chen is presently pursuing his master degree in pharmaceutical analysis in Sun Yat-Sen University. His current research
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focuses on electrochemical sensor and its applications in cancer cell research and drug evaluation.
Yunfei Jiang
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is presently pursuing her master degree in pharmaceutical analysis in Sun Yat-Sen University. Her current
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research focuses on electrochemical sensor and its applications in cancer cell research and drug evaluation.
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Zhiru Wang is presently pursuing her master degree in pharmaceutical analysis in Sun Yat-Sen University. Her current research focuses on electrochemical sensor and its applications in cancer cell research and drug evaluation.
received her Ph.D. degree from Shanghai Institute of Applied Physics, Chinese Academy of Sciences in 2016.
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Weiwei Qin
She is presently employed as a senior research associate in School of Pharmaceutical Sciences, Sun Yat-Sen University. Her current
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research interests are DNA nanotechnology and biosensor.
Yanyan Yu received her Ph.D. degree from Sun Yat-Sen University in 2015. She is presently
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employed as a lecturer in School of Pharmacy, Nantong University. Her current research interests are electrochemical biosensor and DNA nanotechnology. Zuanguang Chen
received his B.S. and Ph.D. degrees in analytical chemistry from Sun Yat-Sen University in 1982 and
2000, respectively. As a professor and director of the Institute of Analytical Instrument, School of Pharmaceutical Sciences, Sun
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Yat-Sen University, his current research interests include analytical instruments, capillary electrophoresis, microfluidic system,
electrochemical sensor, and pharmaceutical analysis.
Yuanqing Zhang received his Ph.D. degree in Shanghai Institute of Applied Physics, Chinese Academy of Sciences in 2011. He worked as a post-doctoral fellow in Houston Methodist Research Institute of United States from 2011 to 2015. As a professor
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multifunctional nanomaterial and their applications in diagnosis, metastasis, and treatment of cancer.
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in School of Pharmaceutical Sciences, Sun Yat-Sen University, his current research interests include microfluidic chip,
Fig. 1. (A) SEM image of the prepared Pd-Pt nanocages. The inset highlights Pd-Pt nanocages. (B) TEM image of Pd-Pt nanocages. The inset highlights one Pd-Pt nanocage. (C) Size 28
distribution of Pd nanocubes synthesized with average diameters of 20 nm, and (D) Pd-Pt
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nanocages with average diameters of 32 nm.
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Fig. 2. (A) Schematic illustration of the aptamer-containing DNA nanotetrahedron from four
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single-stranded DNA sequences and the formation of DNA nanotetrahedron and
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complementary DNA1. (B) Agarose gel electrophoretic analysis of the gradual formation of NTHs. Lane M: DNA marker; Lane 1 is THA only; Lane 2 is THA + THB; Lane 3 is THA +
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THB + THC; Lane 4 is THA + THB + THC + THD; Lane 5 is THA + THB + THC + THD +
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cDNA1.
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Fig. 3. (A) DPV responses of the nanoprobe1/MCH/aptamer-NTH/SPGE in the (a) PBS solution (100 mM, pH 7.0), (b) PBS solution with only 3 mM HQ, and (c) PBS solution with 3 mM HQ and 2 mM H2O2. (B) DPV responses of the (a) cDNA1-HRP-Pd-Pt nanocages/MCH/aptamer-NTH/SPGE, (b) nanoprobe1/MCH/aptamer-NTH/SPGE, and (c)
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nanoprobe1+nanoprobe2/MCH/aptamer-NTH/SPGE in the PBS solution (100 mM, pH 7.0)
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M
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with 3 mM HQ and 2 mM H2O2.
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Fig. 4. (A) DPV responses with different concentrations of HepG2 cells (from a to f: 10, 102, 103, 104, 105, and 106 cells mL-1). (B) The resulting calibration curve of the logarithm value of the cell concentration vs peak current. (C) DPV responses and (D) the corresponding histogram obtained with various types of cells with the same concentration and their mixture solution. 30
Cell concentration: 105 cells mL-1. The error bars represent the standard deviations of three
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parallel determination results.
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Fig. 5. (A) Fluorescent microscope images of HepG2 cells stained by calcein-AM after
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captured onto the screen-printed gold electrodes surface. (B) The cell viability of the released
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HepG2 cells with different electrochemical potential. The error bars represent the standard
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deviations of three parallel determination results.
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Scheme 1. (A) Schemes illustrating the major steps for the fabrication of Pd-Pt nanocage–
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HRP–cDNA/hemin/G-quadruplex hybrid nanoprobes. (B) Schemes illustrating the prepared process of the proposed electrochemical aptasensor for the capture, detection, release of CTCs.
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(CE: counter electrode, WE: working electrode, RE: reference electrode)
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Table 1. Comparison of this electrochemical aptasensor with other reported cytosensors for HepG2 cell detection Detection Immobilized Cytosensor type
Linear range
Analytical method
Referenc limit
-1
receptor
(cells mL )
e -1
Electrochemiluminescenc
nt immunosensor
e
3×102 to
Anti-EpCAM antibody
1×10
4
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Electrochemiluminesce
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(cells mL )
Microcantilever assay
TLS11a aptamer
1×10
Anti-EpCAM
plasma mass
antibody and TLS11a
spectrometry
aptamer
N
Inductively coupled
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Magnetic
Electrochemical
M
immunosensor
Galactosylated gold
40 to 8×103
[41]
15
[42]
30
[43]
10
[14]
1×102 to
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Impedance spectroscopy cytosensor
nanoisland
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TLS11a aptamer
voltammetry
Differential pulse
1×10
5
1×102 to
Differential pulse
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Electrochemical
1×10
7
TLS11a aptamer-
This 6
10 to 1×10 voltammetry
DNA nanotetrahedron
A
aptasensor
300
5
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aptasensor
Electrochemical
[40]
1×103 to
Microcantilever
aptasensor
256
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5 work
S0925-4005(18)30846-3 https://doi.org/10.1016/j.snb.2018.04.142 SNB 24618
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
9-1-2018 20-4-2018 24-4-2018
Please cite this article as: Duanping Sun, Jing Lu, Dabin Chen, Yunfei Jiang, Zhiru Wang, Weiwei Qin, Yanyan Yu, Zuanguang Chen, Yuanqing Zhang, Label-free electrochemical detection of HepG2 tumor cells with a self-assembled DNA nanostructure-based aptasensor, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.04.142 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.
Label-free electrochemical detection of HepG2 tumor cells with a self-assembled DNA nanostructure-based aptasensor
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Duanping Suna,1, Jing Lua,1, Dabin Chena,1, Yunfei Jianga, Zhiru Wanga,
a
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Weiwei Qina, Yanyan Yub, Zuanguang Chena,*, Yuanqing Zhanga,*
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, Guangdong
School of Pharmacy, Nantong University, Nantong, Jiangsu 226001, China
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* Corresponding authors.
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b
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510006, China
1
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(Y. Zhang).
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E-mail addresses: [email protected] (Z. Chen), [email protected]
These authors contributed equally to this work.
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Graphical Abstract
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Highlights
Aptasensor was developed for the electrochemical detection and release of CTCs. Tetrahedron-based aptamer was used for the highly enhanced capture of
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target cells.
Dendritic structure nanoprobes amplified the electrochemical signals
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significantly.
The detection limit of the cytosensor could be estimated as 5 cells mL−1 for
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This method released the captured HepG2 cells with little damage for further
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HepG2.
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study.
Abstract
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The development of rapid, sensitive and convenient methods for the monitoring of rare circulating tumor cells (CTCs) is of great significance in cancer diagnostics and therapy.
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Herein a label-free and competitive electrochemical aptasensor was proposed for the efficient capture, ultrasensitive detection and controlled release of CTCs. Firstly, the DNA nanotetrahedron (NTH)-based TLS11a aptamer probe was immobilized on a disposable screen-printed gold electrode (SPGE) surface via the Au−S bonds for the 2
highly enhanced capture of liver cancer HepG2 cells. Then, hybrid nanoprobes of PdPt nanocages labeled with complementary DNA (cDNA), hemin/G-quadruplex DNAzyme and horseradish peroxidase (HRP) were attached on the SPGE substrate by the DNA hybridization, resulting in the formation of dendritic structure (DS)
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nanoprobes with self-assembly methods for the greatly enhanced sensitivity. When the target HepG2 cells existed, they can compete with DS nanoprobes to bind with NTH-
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based aptamer probe, resulting in the release of the DS nanoprobes from the SPGE. This method exhibits ultrahigh selectivity and sensitivity toward HepG2 with detection
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limits of 5 cells per ml. Furthermore, our strategy allows for easy detachment of the
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captured cells from the SPGE without compromising cell viability by an
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electrochemical cleavage of the Au–S bonds. The present study provides a label-free
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technique for highly selective and efficient quantification of tumor cells, which is
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essential in the early cancer diagnosis and treatment of cancer. Keywords: electrochemical cytosensor; aptamer, HepG2 cells; nanotetrahedron;
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dendritic structure nanoprobes
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1. Introduction
Cancer is a highly fatal disease and has become one of the biggest killers of human
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health in the world [1-3]. Circulating tumor cells (CTCs), a type of cancer cells that can circulate in the peripheral blood or through the lymphatic system, can lead to a new tumor metastasis. Numerous clinical studies has indicated that CTCs can act as a biomarker in the early detection of cancer, cancer diagnosis and therapy assessment 3
[4,5]. However, the number of tumor cells circulating in human blood are exceedingly rare (1–3000 CTCs per mL against 109 red blood cells per mL) [6]. Thus there is an urgent demand to capture and detect these rare tumor cells accurately. Up to now, various strategies have been designed to determine the cancer cells,
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including fluorescent measurements or imaging [7], flow cytometry [8], magnetic immunoassay [9], electrochemiluminescence [10,11], and electrochemistry [12-17].
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Among them, electrochemical biosensor, especially aptamer-based biosensor
(aptasensor), has received particular attention in cancer cell research due to the intrinsic
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advantages in terms of excellent portability, rapid response and sensitive recognition
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[18-23]. Aptamers are designed through systematic evolution of ligands by exponential
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enrichment (SELEX) system in vitro and single-stranded nucleic acids with specific
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three dimensional structure. They have high specificity and affinity toward the target
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molecules ranging from small molecules like proteins to whole cells [24]. However, because of the flexibility of single-stranded nucleic acids, it tends to undergo self-
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assembled monolayer aggregation by immobilizing aptamers on substrates, which largely impede accessibility for the target tumor cells. It is also difficult to control the
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spatial orientation of single-stranded nucleic acids with precision [21]. As a result, the analytical selectivity and sensitivity is not high enough for cancer cells and it fail to
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preserve accuracy when challenged with a clinical sample. Therefore it is highly desirable to develop a reliable and rapid platform to analysis tumor cells specifically and sensitively with modest requirements of sample volumes. To enhance the selectivity and sensitivity of electrochemical aptasensor, various 4
methods have been employed to modify working electrode [25-27]. DNA nanotechnology is emerging as a novel nanotechnology with several unique advantages including a highly programmable nature, ease of preparation and high precision. On the basis of these, DNA nanotetrahedron (NTH) is a classical three-dimensional framework,
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which was originally reported by Turberfield and co-workers [28]. Due to the powerful structural rigidity and excellent biocompatibility, DNA NTH can be an ideal strategy
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for the electrochemical aptasensing platform [29, 30]. For example, Fan and colleagues
developed a series of electrochemical biosensors based on self-assembled DNA NTH
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nanostructures for greatly increasing target accessibility and improving the sensitivity.
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As a consequence of the attractive features of NTH, DNA tetrahedron-based aptamer
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probe can provide a rigid scaffold for cancer cell recognition with high selectivity and
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high reproducibility [31,32]. However, only by using DNA NTH-based aptamer for
clinical samples.
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electrochemical cytosensing [21], the detection sensitivity is not good enough for the
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In addition, through the DNA hybridization with layer-by-layer (LBL) assembly process, dendritic structure (DS) DNA is an excellent strategy for immobilizing
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abundant signal tags, nanomaterials or biomolecules, such as nature enzymes (peroxidase and glucose oxidase) and mimic enzymes (hemin/G-quadruplex DNAzyme)
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[33]. Meanwhile, because of the excellent electrocatalytic capacity through in situ forming the DS biomolecules or nanomaterials, DSDNA provides a very favorable method for electrochemical signal amplification [34]. As a consequence of the excellent stability, DSDNA has garnered particular attention in designing electrochemical 5
aptasensors [35]. Due to the DSDNA with enzymatic amplification, we can successfully achieve the efficient improvement of detection sensitivity of the electrochemical aptasensing platforms for tumor cells. Screen-printing is one of the most important methods towards rapid, simple and inexpensive production of electrochemical
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biosensors. Screen-printed electrodes with speed of mass production and low cost have often been used for developing electrochemical biosensors and improving their
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performances [36]. Their main advantage is the reduction of sample volume required,
which in turn helps in reducing the overall size of the biosensing platform into which
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the device will be integrated. Furthermore, SPEs can avoid some of the common
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questions of classical solid electrodes, such as tedious cleaning processes and memory
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effects.
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Herein, we for the first time combined the strengths of advanced aptamer strategy,
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DNA-based nanotechnology and disposable screen-printed electrodes to develop a label-free and competitive electrochemical aptasensor for the capture, detection and
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release of HepG2 tumor cells (Scheme 1). Firstly, we incorporated an expanded nucleotide-containing aptamer into a DNA NTH structure for greatly enhanced capture
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of cancer cells. Then the self-assembled NTH-based TLS11a aptamer probe [13,22] was immobilized on a disposable screen-printed gold electrode (SPGE) surface via
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Au−S interactions. Nanoprobes (nanoprobe1 and nanoprobe2) were fabricated by PdPt nanocages labeled with two complementary DNA (cDNA1 and cDNA2), horseradish peroxidase (HRP), and hemin/G-quadruplex DNAzyme (Scheme 1A). The nanoprobes were captured onto the SPGE surface via the DNA hybridization, resulting in the 6
formation of DS nanoprobes for the greatly enhanced sensitivity. When the target cells existed, NTH-based TLS11a aptamer probe efficiently captured the target HepG2 cells, resulting in the denaturation of double-stranded DNA and the release of the DS nanoprobes. Finally, captured HepG2 cells can be released from the SPGE for further
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analysis by an electrochemical cleavage of the Au–S bonds via a negative potential (Scheme 1B). In this way, the proposed electrochemical aptasensor showed excellent
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selectivity and sensitivity, indicating potential applications in the early diagnosis and treatment of cancers and choice of individual sensitive anti-cancer drugs.
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Scheme 1
(HPLC
purified)
were
synthesized
from Sangon
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All oligonucleotides
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2.1. Materials and apparatus
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2. Experimental section
Biotechnology Co., Ltd. (Shanghai, China) with/without thiol labeling. The sequences
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of the oligonucleotides were listed in Table S1 (Supplementary material). Other detailed chemicals or instruments are provided in
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the Supplemental material. All reagents were of analytical grade and were used without further purification. All solutions were prepared with ultrapure water (18.2 MΩ∙cm,
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Milli-Q, Millipore). The electrochemical detection was performed on disposable screen-printed gold electrode (SPGE) with 4 mm diameter working area (DropSens220AT, LLanera, Spain). 2.2. Blood sample preparation 7
Different number of HepG2 cells were spiked into the blood, which was collected from healthy volunteers. The whole blood samples were centrifuged at 1500 rpm for 5 min, then the supernatant was discarded. After this step, 1 ml of red blood cell lysis buffer were added into blood, and incubated at room temperature for 5 min. Finally, the
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sediment was washed and resuspended in PBS solution to obtain a cell suspension. 2.3. Preparation of nanoprobes
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According to the reported method [35,37], Pd-Pt nanocages were prepared.
Detailed steps are provided in the Supplemental materials. 50 μL cDNA1 (5 μM) or
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cDNA2 (5 μM) added in 1 mL Pd-Pt nanocages was stirred for 12 h at 4 °C.
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Subsequently, 50 μL of HRP (1 mg mL-1) were added to the solution and stirred for 6 h
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to form nanoprobes of cDNA1-HRP-Pd-Pt nanocages or cDNA2-HRP-Pd-Pt
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nanocages. Then hemin (0.1 mg) was added to the solution (pH 7.0) for 2 h at 4 °C to
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form bioconjugates of hemin/G-quadruplex DNAzyme. After the supernatant was discarded, the nanoprobe of hemin/G-quadruplex-cDNA1-HRP-Pd-Pt nanocages
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(nanoprobe1) or hemin/G-quadruplex-cDNA2-HRP-Pd-Pt nanocages (nanoprobe2) was obtained and redispersed in 500 mL PBS (10 mM, pH 7.4) solution at 4 °C for
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further use.
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2.4. Preparation of nanotetrahedra The nanotetrahedra were assembled from the three thiolated DNA strands of 61
nucleotides (THA, THB, and THC) and one aptamer-containing DNA strand of 125 nucleotides (THD). Before the assembly of nanotetrahedra, the monomers (THA, THB, THC, and THD) were pretreated with 10 mM TCEP for 2 h at room temperature (RT) 8
to cleave the disulfide linkage. Then the DNAs were mixed equivalently in TM buffer, heated at 95 °C for 10 min, and rapidly cooled at 4 °C for 2 min. The formation of DNA nanostructures was analyzed using 3% agarose gel electrophoresis in TBE buffer at 100 V for 25 min.
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2.5. Fabrication of electrochemical aptasensor The fabrication process of the proposed aptasensor for HepG2 cells was shown in
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Scheme 1B. 10 μL of 500 nM NTH-based TLS11a aptamer probe was immobilized on
the surface of SPGE at RT for 2 h, and the unbound aptamer probes were thoroughly
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washed away with ultrapure water. After immobilization, the blank sites on the SPGE
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were then blocked with 1 mM 6-mercapto-1-hexanol (MCH) for 2 h. Unbound MCH
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was also washed away with ultrapure water. Finally, 10 μL nanoprobe1 and 10 μL
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nanoprobe2 were dropped onto the obtained SPGE surface for 2 h via the hybridization
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of aptamer and cDNA1, resulting in the formation of DS nanoprobes via the hybridization of cDNA1 and cDNA2. Each step was characterized by differential pulse
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voltammetry (DPV). As a control, only 10 μL nanoprobe1 were dropped on the SPGE surface and incubated under the same conditions.
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2.6. Cell capture and electrochemical detection A 200 μL HepG2 cells with a certain concentration in PBS solution was dropped
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on the modified SPGE and incubated at 37 °C for 1 h to capture the target cells. Then the SPGE was carefully washed with 10 mM PBS for three times. To determine the cell viability, the captured cells on the SPGE were stained with calcein acetoxymethyl ester (calcein-AM, a cell viability indicator). As a control, a 200 μL of PBS buffer containing 9
MCF-7 cells and a 200 μL 1:1 mixture of MCF-7 cells and HepG2 cells were dropped on the SPGE surface and incubated under the same conditions. For electrochemical detection, the SPGE was placed in 3 mL of 100 mM PBS (pH 7.0) solution containing 3 mM hydroquinone (HQ) and 2.0 mM H2O2. DPV analysis was performed from 0.2 to
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−0.3 V at 50 mV pulse amplitude. 2.7. Cell release and viability analysis
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After electrochemical detection, a negative potential was performed to break Au–
S bonds on the SPGE to release the captured HepG2 cells. The cyclic voltammetry (CV)
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was carried out in the PBS (10 mM, pH 7.4) solution, ranging from -0.9 to -1.7 V at a
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scan rate of 50 mV s-1. Released cells were observed and counted using a microscope
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system. Furthermore, cell viability were determined by trypan blue staining assay. After
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the cells were released, the remaining cells solution were recollected by centrifugation
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for 5 min at 1000 rpm, and redispersed in PBS (10 mM, pH 7.4) for staining assay experiment. The released cells were immersed into a trypan solution (0.04%) for 3 min.
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By trypan blue staining, viable cancer cells and dead cancer cells can be observed using
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a microscope system.
3. Results and discussion
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3.1. Principle of the cytosensor for HepG2 detection The self-assembled DNA nanostructure-based electrochemical aptasensor for capturing, detecting and releasing of tumor cells was illustrated in Scheme 1. In this work, Pd-Pt nanocages, with HRP-mimicking catalytic activity and high surface area, 10
were used as the nanocarriers for loading the amount of biomolecules [35, 37]. The cDNA consisted of two sequences (DNA hybridization sequences and G-quadruplexforming sequences, as shown in Table S1). In the presence of hemin, the catalytic Gquadruplex/hemin DNAzymes could be formed. G-quadruplex/hemin DNAzymes, a
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complex between hemin and a single-stranded guanine-rich nucleic acid, has been widely used as HRP-mimicking DNAzymes electrocatalysts for electrochemical
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cytosensing [12-14]. The nanoprobe involves the immobilization of complementary DNA (cDNA), hemin/G-quadruplex DNAzyme and HRP on the surface of Pd-Pt
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nanocages through a LBL assembly process (Scheme 1A). The signal amplification
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strategy of the electrochemical cytosensor was depended on the electrochemical feature
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of benzoquinone (BQ) produced from the oxidation of HQ with H2O2, which was
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catalyzed by Pd-Pt nanocages, HRP [38, 39] and HRP-mimicking G-quadruplex/hemin
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DNAzyme together. The process is shown as below H2O2 + H2Q → Q + 2H2O
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Q + 2H+ + 2e- → H2Q
where H2Q is hydroquinone and Q is benzoquinone.
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Then, we incorporated an expanded nucleotide-containing TLS11a aptamer into a DNA NTH structure to modify working electrode of SPGE for enhanced
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electrochemical analysis of CTCs. Nanoprobe1 can be attached on the SPGE through the hybridization of TLS11a aptamer and cDNA1. Meanwhile, nanoprobe1 and nanoprobe2 can form the DS nanoprobes by LBL assembly with the hybridization of cDNA1 and cDNA2. A large amount of HRP, HRP-mimicking DNAzyme and 11
nanocages of the formed DS nanoprobes can significantly amplify the electrochemical signal and improve the sensitivity. When the target cancer cells existed, NTH-based TLS11a aptamer probe can recognize and capture the target HepG2 cells. And it can result in the denaturation of double-stranded DNA structure and the release of the DS
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nanoprobes from the surface of SPGE. Finally, by using the electrochemical method (negative potential), the captured cancer cells were released from SPGE surface by
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cleaving the Au-S bonds on the SPGE surface, resulting in the further analysis of the
tumor cells (Scheme 1B). As a result, the selectivity and sensitivity of the
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electrochemical aptasensor is greatly enhanced based on the DNA NTH structure,
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3.2. Characterization of Pd-Pt nanocages
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aptamer method and the DSDNA strategy.
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The morphology of the prepared Pd-Pt alloy nanocages was characterized using
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scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. As can be seen from Fig. 1A, it is clear that the formed Pd-Pt nanocages showed
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cubic shape and coarse surface. And we also observed a darker contrast in the center, indicating the nanocages with porous walls and hollow interiors, as shown in Fig. 1B.
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From Fig. 1C and D, the formed Pd-Pt nanocages exhibited a slightly larger size than that of the Pd nanocubes. The diameters of the nanocages were uniformly distributed at
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approximately 32 nm (Fig. 1D) and average edge length of Pd nanocubes was about 20 nm (Fig. 1C). These results demonstrated the successful synthesis of Pd-Pt nanocages, and the morphology was acceptable compared to those reported [35, 37]. Figure 1 12
3.3. Characterization of aptamer and DNA nanotetrahedron (NTH) structure The TLS11a aptamer (Fig. S1) was selected for HepG2 cells, which can specifically bind to the protein expressed on the surface of human HepG2 cells [12,22].
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To allow a more direct visualization of TLS11a aptamer and HepG2 cells, fluorescence microscopy was performed to investigate HepG2 cells incubated with fluorescein
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isothiocyanate (FITC)-labeled TLS11a aptamer. As can be seen from Fig. S2, FITCTLS11a aptamer labeled HepG2 cells showed green fluorescence, providing solid
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evidence of the good binding ability of aptamer TLS11a to HepG2 tumor cells.
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As shown in Fig. 2A, NTHs, self-assembled from four single-stranded sequences,
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were introduced to enhance detection performance in this work. Briefly, TLS11a
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aptamers are built into one of the NTH sequences (THD), and the other three (THA,
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THB and THC) end with thiol groups were attached to the SPGE surface via Au−S interactions. Then the hybridization of with aptamer-based NTH and cDNA1 would
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result in the formation of new NTH structure. As shown in Fig. 2B, agarose gel electrophoretic method was performed to
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demonstrate our NTH structure. The NTH (lane 4) migrated more slowly than combinations constructed of fewer than four single-stranded sequences (lane 1, lane 2
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and lane 3). And the only major clear band can be observed on the gel, because of the rapid and specific DNA hybridization ensuring the high yield of NTHs. By observing lane 5 (the DNA hybridization of aptamer-based NTHs and cDNA1), a new and slower band, compared with that of the NTHs (lane 4), could be seen. In order to further 13
investigate the behavior of TLS11a, cDNA1 and cDNA2, agarose gel characterization was also performed. With the addition of strands from lane 1 to lane 3, it is clear that a significant reduction of electrophoretic mobility could be observed, which can be attributed to the increased molecular mass (Fig. S3). Therefore, the results
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demonstrated that our aptamer-based NTHs had been successfully formed and its response to the HepG2 tumor cells was further studied in the following electrochemical
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experiments.
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3.4. Electrochemical characterization of cytosensor
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Figure 2
A
To understand the events occurring on the SPGE surface, CV and EIS
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characterization were performed. Fig. S4A and B shows the CV and EIS curves of the
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modified SPGE in the assembly processes. For the bare SPGE, a couple of reversible redox peaks were observed (curve a, Fig. S4A) and the Nyquist plot included no
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semicircle domain (curve a, Fig. S4B). However, after the modification of NTH-based aptamer, the gap between the cathodic and anodic peaks became wider and the peak
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current decreased obviously (curve b, Fig. S4A); meanwhile, a small semicircle was observed (curve b, Fig. S4B), suggesting the nonconductive DNA blocked the electron
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transfer. With further the modification of MCH, the peak current continuously decreased (curve c, Fig. S4A) and the semicircle gradually increased (curve c, Fig. S4B). After incubating with the nanoprobes, a larger decrease in the peak currents and a larger increase of the semicircle diameter could be seen (curve d, Fig. S4A and S4B), 14
indicating the bad conductivity of the nanoprobes. Finally, the aptasensor was incubated with the HepG2 cells, and the large amount of cancer cells still contributed to a decrease in the peak currents and a much larger semicircle (curve e, Fig. S4A and S4B). These
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results demonstrated the successful assembly of the electrochemical aptasensor.
3.5. Electrochemical characterization of nanoprobes
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Additionally, DPV characterization was utilized to demonstrate the signal amplification effect of the designed nanoprobes. As depicted in Fig. 3A, when the
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nanoprobe1/MCH/aptamer-NTH/SPGE was immersed in the 100 mM pH 7.0 PBS,
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electrochemical responses cannot be observed in the solution (curve a). When the
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nanoprobe1/MCH/aptamer-NTH/SPGE was placed in the 100 mM PBS solution with
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only 3 mM HQ, a weak DPV reduction peak appeared, which was ascribed to the
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electrochemical reduction of HQ (curve b). As can been seen in curve c, a large shift in the DPV peak was observed, when the nanoprobe1/MCH/aptamer-NTH/SPGE was
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placed in the 100 mM PBS solution with 2 mM H2O2 and 3 mM HQ. The results can indicate that the nanoprobes exhibits its good peroxidase catalytic activity by efficiently
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catalyzing the oxidation of HQ with H2O2. As can be seen from Fig. 3B, compared with the peak current of cDNA1-HRP-Pd-
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Pt nanocages (curve a), the nanoprobe1 with the HRP-mimicking DNAzyme, HRP and Pd-Pt nanocages together catalyzed oxidation of the redox-tags (HQ) with H2O2 and amplified the electrochemical signals (curve b, nanoprobe1). However, larger increase of DPV peak current was observed through the formation of the DS nanoprobes (curve 15
c, nanoprobe1 + nanoprobe2). Due to the A-T and G-C base pairs, a large amount of HRP-mimicking DNAzyme, HRP and Pd-Pt nanocages can be embedded into DS nanoprobes. Therefore, the DS nanoprobes effectively amplified the electrochemical signal due to the synergetic catalysis of numerous Pd-Pt nanocages, HRP and HRP-
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mimicking DNAzyme to the redox-tags (HQ) with H2O2.
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Figure 3
3.6. Optimization of the experimental conditions
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To achieve the optimum cell capture time, HepG2 cell solution was dropped onto
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the SPGE surface and incubated for different times (30, 45, 60, 75, 90 min). As shown
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in Fig. S5A, the current signal strengthened along increasing the cell incubation time.
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However, the minimum signal was achieved at 60 min; thus, 60 min was chosen as the
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optimal cell incubation time. We also investigated the concentration of HQ on the analytical performance of the electrochemical aptasensor. Fig. S5B displays the effect
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of the HQ concentration on the DPV responses. The signal gradually increased with increasing HQ concentration from 1.0 to 4.0 mM. With more than 3 mM, the current
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signal reached a plateau. Thus, the optimal HQ concentration was 3 mM in subsequent
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experiments.
3.7. Cell capture and electrochemical detection The high sensitivity plays an important role in the early clinical diagnosis. Under the optimum conditions, the current signal decreased along with the increasing 16
concentration of HepG2 cells in PBS buffer. Fig. 4A displays the DPV current signals after the detection of different concentration of HepG2 cells from 10 to 1×106 cells mL−1. There is a good linear correlation between the logarithm values of HepG2 cell concentration (Ccell) and the DPV current value. The linear equation is I (μA) =37.6896
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– 5.6055 lgCcell (cells mL−1) (R2 = 0.9961), as depicted in Fig. 4B. And the detection limit is calculated as 5 cells mL−1 for HepG2 cells by using 3σ method. The
electrochemiluminescence
immunosensor,
inductively
coupled
plasma
mass
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spectrometry and microcantilever aptasensor (Table 1).
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performance is better than that of previous methods for HepG2 cells, such as
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The high selectivity is another important criterion in the early cancer diagnosis. To
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further investigate the specificity of the aptasensor, breast cancer MCF-7 cells, liver
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cancer HepG2 cells and the mixture of HepG2 cells and MCF-7 cells were tested. As
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shown in Fig. 4C and D, compared with the blank control, no obvious current signal was observed for MCF-7 cells (105 cell mL-1). Moreover, when the aptasensor was performed to detect HepG2 cells and the mixture, there was conspicuous current signal
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for HepG2 cells (105 cell mL-1). The above results demonstrated that the selectivity of
Figure 4
A
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the aptasensor was satisfactory for the determination of target HepG2 cells.
Nest, to understand the repeatability of the aptasensor, relative standard deviations (RSD) were utilized to estimate the repeatability. According to the data from five independently fabricated SPGEs for the same cell concentration (104 cells ml-1), the 17
electrochemical cytosensor performed a RSD of 6.53%, demonstrating the acceptable repeatability of the cytosensor for the analysis of target HepG2 cells. In addition, the stability of the aptasensor was also studied. After storage at 4°C for one week, no obvious change in electrochemical signal was observed, which suggested a satisfactory
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stability of the nanostructured-based cytosensor. Furthermore, to explore the potential clinical application of the aptasensor, we
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spiked blood with various concentrations of HepG2 cells (103 and 105 cell mL−1). As shown in Table S2, the results displays the recovery of 87.3% and 85.1% and the RSD
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of 5.28% and 6.35%, respectively, indicating the good performance of the strategy.
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These results demonstrate that the electrochemical method provides a potential clinic
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diagnostic tool for the analysis of tumor cells in blood sample.
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3.8. Cell release and viability analysis
The successful release of CTCs from substrate without damage plays key role in
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subsequent study of cell analysis [44-46]. In this work, we uses an electrochemical desorption method for the efficient release of HepG2 tumor cells by cleaving the Au-S
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bonds on the SPGE interfaces. Before cell release, there were a large population of alive HepG2 cells on the substrate by observing the fluorescence signals (Fig. 5A). Fig. S6A
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and B shows the electrochemical curves of the original SPGE and regenerated SPGE by applying an electrical potential. Compared with the original SPGE, the regenerated SPGE had the same peak current and impedance spectra. Fig. S6C shows the black fluorescence image of the SPGE surface after cell release. There results suggested a 18
nearly 100% of the captured cells were released by this electrochemical method. To further investigate the cell viability of released cells (the ratio of alive cells to total cells), we treated the SPGE with the different electrochemical potential with -0.9, -1.1, -1.3, -1.5, and -1.7 V for 30s, respectively. The results in Fig. 5B suggested the
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viability decreased to 94% due to the little amount of dead cells. The released HepG2 cells from SPGE by the electrochemical method were further collected, and the
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microscope image was shown in Fig. S6D. The results demonstrates that the released cells still maintain high viability for further study.
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Figure 5
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4. Conclusions
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In conclusion, a label-free, portable and competitive electrochemical cytosensor
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was proposed for the capture, detection and release of HepG2 tumor cells. Compared with the previous methods with DNA tetrahedron-based aptamer [21, 31], we combined
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the strengths of DNA-based nanotechnology and DS nanoprobes (with a relatively large amount of enzyme) for highly efficient capture, ultrasensitive electrochemical detection
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and controlled release of the HepG2 cells in the SPGE. Due to the unique property of the DNA NTH-based aptamer probe and in situ LBL assembly of DS nanoprobes, the
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present method significantly improves selective and sensitive electrochemical performance for analyzing tumor cells. Moreover, the captured cells can be released with little damage by an electrochemical desorption method. The results show the high selectivity, acceptable sensitivity and good reliability. The low detection limit is 5 cells 19
per mL. This proposed strategy provides a powerful tool for comprehensive studies of CTCs and to show the great potential in early cancer diagnosis.
Acknowledgments
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This work was supported by the National Natural Science Foundation of China (No. 81601571 and 21675177), the Science and Technology Planning Project of Guangdong
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Province (No. 2016B030303002) and the Medical Scientific Research Foundation of
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Guangdong Province (No. A2017033).
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found in the online version.
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[45] J. Cao, X.P. Zhao, M.R. Younis, Z.Q. Li, X.H. Xia, C. Wang, Ultrasensitive capture, detection, and release of circulating tumor cells using a nanochannel–ion channel hybrid coupled with electrochemical detection technique, Anal. Chem. 89 (2017) 10957-10964.
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electrochemical release of circulating tumor cells by using aptamers modified gold nanowire
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Biographies Duanping Sun
received his Ph.D. degree in pharmaceutical analysis from Sun Yat-Sen University in 2016 under the
supervision of Professor Zuanguang Chen. He is presently employed as a senior research associate in School of Pharmaceutical
Sciences, Sun Yat-Sen University. His current research interests focus on electrochemical biosensor, microfluidic chip, and
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pharmaceutical analysis.
Jing Lu received her Ph.D. degree from Sun Yat-Sen University in 2017. She is presently employed as a senior research associate
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in School of Pharmaceutical Sciences, Sun Yat-Sen University. Her current research interests include electrochemical biosensor
and pharmacology.
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Dabin Chen is presently pursuing his master degree in pharmaceutical analysis in Sun Yat-Sen University. His current research
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focuses on electrochemical sensor and its applications in cancer cell research and drug evaluation.
Yunfei Jiang
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is presently pursuing her master degree in pharmaceutical analysis in Sun Yat-Sen University. Her current
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research focuses on electrochemical sensor and its applications in cancer cell research and drug evaluation.
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Zhiru Wang is presently pursuing her master degree in pharmaceutical analysis in Sun Yat-Sen University. Her current research focuses on electrochemical sensor and its applications in cancer cell research and drug evaluation.
received her Ph.D. degree from Shanghai Institute of Applied Physics, Chinese Academy of Sciences in 2016.
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Weiwei Qin
She is presently employed as a senior research associate in School of Pharmaceutical Sciences, Sun Yat-Sen University. Her current
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research interests are DNA nanotechnology and biosensor.
Yanyan Yu received her Ph.D. degree from Sun Yat-Sen University in 2015. She is presently
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employed as a lecturer in School of Pharmacy, Nantong University. Her current research interests are electrochemical biosensor and DNA nanotechnology. Zuanguang Chen
received his B.S. and Ph.D. degrees in analytical chemistry from Sun Yat-Sen University in 1982 and
2000, respectively. As a professor and director of the Institute of Analytical Instrument, School of Pharmaceutical Sciences, Sun
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Yat-Sen University, his current research interests include analytical instruments, capillary electrophoresis, microfluidic system,
electrochemical sensor, and pharmaceutical analysis.
Yuanqing Zhang received his Ph.D. degree in Shanghai Institute of Applied Physics, Chinese Academy of Sciences in 2011. He worked as a post-doctoral fellow in Houston Methodist Research Institute of United States from 2011 to 2015. As a professor
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M
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multifunctional nanomaterial and their applications in diagnosis, metastasis, and treatment of cancer.
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in School of Pharmaceutical Sciences, Sun Yat-Sen University, his current research interests include microfluidic chip,
Fig. 1. (A) SEM image of the prepared Pd-Pt nanocages. The inset highlights Pd-Pt nanocages. (B) TEM image of Pd-Pt nanocages. The inset highlights one Pd-Pt nanocage. (C) Size 28
distribution of Pd nanocubes synthesized with average diameters of 20 nm, and (D) Pd-Pt
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nanocages with average diameters of 32 nm.
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Fig. 2. (A) Schematic illustration of the aptamer-containing DNA nanotetrahedron from four
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single-stranded DNA sequences and the formation of DNA nanotetrahedron and
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complementary DNA1. (B) Agarose gel electrophoretic analysis of the gradual formation of NTHs. Lane M: DNA marker; Lane 1 is THA only; Lane 2 is THA + THB; Lane 3 is THA +
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THB + THC; Lane 4 is THA + THB + THC + THD; Lane 5 is THA + THB + THC + THD +
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cDNA1.
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Fig. 3. (A) DPV responses of the nanoprobe1/MCH/aptamer-NTH/SPGE in the (a) PBS solution (100 mM, pH 7.0), (b) PBS solution with only 3 mM HQ, and (c) PBS solution with 3 mM HQ and 2 mM H2O2. (B) DPV responses of the (a) cDNA1-HRP-Pd-Pt nanocages/MCH/aptamer-NTH/SPGE, (b) nanoprobe1/MCH/aptamer-NTH/SPGE, and (c)
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nanoprobe1+nanoprobe2/MCH/aptamer-NTH/SPGE in the PBS solution (100 mM, pH 7.0)
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M
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with 3 mM HQ and 2 mM H2O2.
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Fig. 4. (A) DPV responses with different concentrations of HepG2 cells (from a to f: 10, 102, 103, 104, 105, and 106 cells mL-1). (B) The resulting calibration curve of the logarithm value of the cell concentration vs peak current. (C) DPV responses and (D) the corresponding histogram obtained with various types of cells with the same concentration and their mixture solution. 30
Cell concentration: 105 cells mL-1. The error bars represent the standard deviations of three
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parallel determination results.
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Fig. 5. (A) Fluorescent microscope images of HepG2 cells stained by calcein-AM after
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captured onto the screen-printed gold electrodes surface. (B) The cell viability of the released
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HepG2 cells with different electrochemical potential. The error bars represent the standard
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deviations of three parallel determination results.
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Scheme 1. (A) Schemes illustrating the major steps for the fabrication of Pd-Pt nanocage–
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HRP–cDNA/hemin/G-quadruplex hybrid nanoprobes. (B) Schemes illustrating the prepared process of the proposed electrochemical aptasensor for the capture, detection, release of CTCs.
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(CE: counter electrode, WE: working electrode, RE: reference electrode)
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Table 1. Comparison of this electrochemical aptasensor with other reported cytosensors for HepG2 cell detection Detection Immobilized Cytosensor type
Linear range
Analytical method
Referenc limit
-1
receptor
(cells mL )
e -1
Electrochemiluminescenc
nt immunosensor
e
3×102 to
Anti-EpCAM antibody
1×10
4
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Electrochemiluminesce
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(cells mL )
Microcantilever assay
TLS11a aptamer
1×10
Anti-EpCAM
plasma mass
antibody and TLS11a
spectrometry
aptamer
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Inductively coupled
A
Magnetic
Electrochemical
M
immunosensor
Galactosylated gold
40 to 8×103
[41]
15
[42]
30
[43]
10
[14]
1×102 to
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Impedance spectroscopy cytosensor
nanoisland
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TLS11a aptamer
voltammetry
Differential pulse
1×10
5
1×102 to
Differential pulse
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Electrochemical
1×10
7
TLS11a aptamer-
This 6
10 to 1×10 voltammetry
DNA nanotetrahedron
A
aptasensor
300
5
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aptasensor
Electrochemical
[40]
1×103 to
Microcantilever
aptasensor
256
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5 work