Author’s Accepted Manuscript

A novel cytosensor based on Pt@Ag nanoflowers

and AuNPs/Acetylene black for ultrasensitive and
highly specific detection of Circulating Tumor
Cells

Sitian Tang, Huawei Shen, Yixiong Hao, Zhenglan

Huang, Yiyi Tao, Yang Peng, Yongcan Guo,
Guoming Xie, Wenli Feng www.elsevier.com/locate/bios

PII: S0956-5663(18)30009-5
DOI: https://doi.org/10.1016/j.bios.2018.01.001
Reference: BIOS10194
To appear in: Biosensors and Bioelectronic
Received date: 8 November 2017
Revised date: 30 December 2017
Accepted date: 3 January 2018
Cite this article as: Sitian Tang, Huawei Shen, Yixiong Hao, Zhenglan Huang,
Yiyi Tao, Yang Peng, Yongcan Guo, Guoming Xie and Wenli Feng, A novel
cytosensor based on Pt@Ag nanoflowers and AuNPs/Acetylene black for
ultrasensitive and highly specific detection of Circulating Tumor Cells,
Biosensors and Bioelectronic, https://doi.org/10.1016/j.bios.2018.01.001
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 A novel cytosensor based on Pt@Ag nanoflowers and

AuNPs/Acetylene black for ultrasensitive and highly specific

detection of Circulating Tumor Cells

Sitian Tanga, Huawei Shenb, Yixiong Haoa, Zhenglan Huanga, Yiyi Taoa,

Yang Penga, Yongcan Guoc, Guoming Xiea1, Wenli Fenga*

a
Key Laboratory of Medical Diagnostics of Ministry of Education,

Department of Laboratory Medicine, Chongqing Medical University,

Chongqing, 400016, P R China

b
Traditional Chinese Medicine Hospital of Chongqing, Chongqing,

400021, P R China
c
Clinical Laboratory of Traditional Chinese Medicine Hospital Affiliated

to Southwest Medical University, Luzhou, 646000, P R China

*
Correspondence authors: Key Laboratory of Medical Diagnostics of

Ministry of Education, Department of Laboratory Medicine, Chongqing

Medical University, No. 1 Yi Xue Yuan Road, Chongqing, 400016, P R

1
Key Laboratory of Medical Diagnostics of Ministry of Education,

Department of Laboratory Medicine, Chongqing Medical University, No.

1 Yi Xue Yuan Road, Chongqing, 400016, P R China,

guomingxie@cqmu.edu.cn, Tel.: +86 23 68485240, fax.: +86 23

68485239
China fengwlcqmu@sina.com Tel.: +86 23 68485938, fax.: +86 23

68485239

Abstract:

Circulating tumor cells (CTCs), as the cellular origin of metastasis,

are cancer cells that break away from a primary tumor and circulate in the

peripheral blood. And they provide a wealth of information about tumor

phenotype. Here, this work reported a novel ultrasensitive immunoassay

protocol for the detection of CTCs by using Pt@Ag nanoflowers

(Pt@AgNFs) and AuNPs/Acetylene black (AuNPs/AB) nanomaterial. In

the established approach, AuNPs/AB nanomaterial was used as substrate

material to increase the specific surface area and enhance the conductivity

of the gold electrode. Protein G was used for oriented immobilization of

capture antibody, which strongly improved the capture efficiency of

MCF-7 cells. The innovatively synthesized Pt@AgNFs by our group with

high specific surface area and good biocompatibility were not only as the

carriers of signal antibodies (Ab2) but also catalyzed the reduction of

H2O2, which effectually amplified the current signal. A linear relationship

between current signals and the concentrations of CTCs was obtained in

the range from 20 to 1×106 cells mL-1 and the detection limit is as low as
3 cells mL-1 on condition of acceptable stability and reproducibility.

Furthermore, the as-proposed cytosensor showed excellent performance

in the detection of CTCs in human blood samples. These results suggest

that the proposed cytosensor will be a promising application for

accurately quantitative detection of CTCs.

Keywords:

Circulating tumor cells; Acetylene black; Pt@Ag nanoflowers;

Nanomaterials; Electrochemical cytosensor

1. Introduction

It is commonly acknowledged that the major cause of

cancer-associated mortality is tumor metastasis. The early monitoring of

cancer cells metastasis from blood is of great clinical significance for

both diagnosis and treatment. As the role of “tumor liquid biopsy”,

circulating tumor cells (CTCs) provide access to some disease sites,

including that of the primary tumor and the site of fatal metastases

(Chaffer and Weinberg 2011; Goldkorn et al. 2014; Rack et al. 2014;

Shen et al. 2017). So, it is conceivable that detecting and analyzing CTCs

will provide insightful information in assessing the disease status, and it

hasn’t the flaws and limitations encountered in performing conventional

tumor biopsies (Park et al. 2011; Song et al. 2017).

Currently, various techniques have been developed for CTCs

detection, including cytological testing, fluorescent imaging, magnetic

resonance imaging, computerized tomography, X-ray radiography and

ultrasound (Brindle 2008; Fass 2008; Zhang et al. 2002). However, there

are various drawbacks in these methods, such as high cost,

time-consuming, low sensitive, lack of specificity, or the need for

professional operators (Shen et al. 2016), which limit the clinical

application. Therefore, the development of a convenient, economical,

accurate CTCs detection technology is urgently needed.

In recent years, electrochemical cytosensor has been a particularly

attractive tool for cancer metastasis monitoring due to its fast response,

low detection limits, high sensitivity, ease of operation and low

manufacturing cost (Giaever and Keese 1993; Hong et al. 2011; Liu et al.

2011; Seriburi et al. 2008; Wu et al. 2017). For instance, an integrated

multifunctional platform based on biotin-doped conducting polymer

nanowires was utilized for detection of MCF-7 cells and HCT-116 cells

(Hong et al. 2016). A sensitive electrochemical aptamer cytosensor based

on the hybrid nanoelectrocatalysts and enzyme was fabricated for highly

specific detection of HepG2 cells (Sun et al. 2016). And

peptide-functionalized nanomaterials were employed for the efficient

isolation and detection of HER2-positive CTCs (Peng et al. 2017).

Although their performances are quite excellent, the signal couldn’t

satisfy the desired sensitivity for the early clinical diagnosis. So, it is an

urgent challenge to develop an efficient signal amplification strategy to

achieve high sensitivity for the cytosensor.

With the rapid development of nanotechnology, a variety of

nanomaterials have been used in the construction of electrochemical

biosensors for the sensitivity enhancement and signal amplification (Wan

et al. 2014; Yang et al. 2016b; Zhang et al. 2015). Carbon nanomaterials

are of great interest in many applications because of their high specific

surface area, good electrical conductivity and large adsorption capacities

(Liang et al. 2008). Among them, a special type of carbon material,

namely, acetylene black (AB), has been intensively studied for their

potential applications in biosensing area because of its excellent electric

conductivity and large specific surface area. These properties of AB are

beneficial in improving the sensitivity of electrochemical detection (Deng

et al. 2011; Wei et al. 2016; Zhou et al. 2016). AuNPs with good

biocompatibility are mostly recommended owing to the fact that they can

greatly increase the current response of the modified electrode with a

good conductive ability and provide binding sites for subsequent protein

(Huo et al. 2016). Based on their excellent properties mentioned above, in

this study, AuNPs/AB has been prepared as a novel functional material

for application, which can obviously improve the sensitivity of the

cytosensor.

Protein-mediated synthesis of inorganic nanomaterials for the

sensing interfaces of electrochemical cytosensors is attractive for a

number of reasons: the synthetic conditions are mild, near room

temperature, in aqueous solutions, and at neutral pH; the sizes, shapes,

morphologies, and crystal structures are complex but controllable; the

resulting products are multifunctional and biocompatible (Cao et al. 2015;

Huang et al. 2011). Ag nanoflowers (AgNFs) which have larger specific

surface areas, excellent electrical conductivity, and especially splendid

horseradish peroxidase activity, provide an outstanding electrochemical

sensing matrix in the construction of cytosensor (Huang et al. 2016; Su et

al. 2010; Zuo et al. 2015). In view of their excellent performance, our

group innovatively adhere Pt nanoparticles (PtNPs) onto the surface of

AgNFs for the novel Pt@AgNFs to further improve the ability for the

reduction of H2O2.

In this paper, a novel cytosensor is fabricated for ultrasensitive

detection of CTCs (MCF-7) based on the Pt@AgNFs and AuNPs/AB

nanomaterial. As shown in Scheme 1, in the cytosensor, AB was firstly

modified onto the Au electrode as advanced electronic materials for

improving the electron-transfer ability towards the electrode surface.

Moreover, it could increase the electrode surface area and thus lead more

AuNPs modified onto the surface, which possessed the ability to anchor
more protein G to amplify the signals. In view of the above two kinds of

nanomaterials, the electrical conductivity of the electrode surface

increased almost 5 times. Protein G was immobilized on the AuNPs for

oriented immobilization of capture antibody (Ab1). It contributed that the

distribution of Ab1 was more uniform and expose more effective binding

sites. At the same time, we use the in-situ synthesis method to adhere

PtNPs onto the surface of AgNFs to form the novel Pt@AgNFs, which

was used as the label of secondary antibodies (Ab2) for the reduction of

H2O2 to amplify the electrochemical signal of the cytosensor. The

proposed cytosensor showed a wide linear range with a greatly low

detection limit. Therefore, the proposed cytosensor has a great prospect in

the early diagnosis of cancer.

2. Experimental

2.1. Reagents and materials

MCF-7 and HeLa cell lines were obtained from the Key Laboratory

of Medical Diagnostics of Ministry of Education and College of Basic

Medicine, Chongqing Medical University. Protein G, bovine serum

albumin (BSA), ascorbic acid (AA), chitosan (CS) and Sodium citrate

were purchased from Sangon Biotech Co. (Shanghai, China). Mouse

monoclonal antibody to EpCAM (capture antibody and signal antibody)

was ordered from Abcam Co. (Shanghai, China). Acetylene black (AB)

was purchased from Yi Huan Carbon Co. (Fuzhou, China). Silver nitrate

(AgNO3), chloroplatinic acid (H2PtCl6·6H2O), chloroauric acid (HAuCl4),

N-hydroxy-succinimide (NHS), 1-ethyl-3- (3-dimethylaminopropyl)

carbodiimide (EDC) were obtained from Sigma-Aldrich (St. Louis, MO,

USA). Deionized distilled water purified through a Millipore system (Z18

MΩ cm) was employed in all runs. All other chemicals were of reagent

grade and used as received.

2.2. Apparatus and instruments

All electrochemical measurements were performed in an

electrochemical workstation (CHI-660E, Shanghai, China). A

conventional three component electrochemical cell, with a modified Au

electrode as the working electrode, a KCl-saturated Ag/AgCl electrode as

the reference electrode, and a Pt wire as the auxiliary electrode, was

employed in all electrochemical measurements. EIS and CV

measurements were used to characterize the interface properties of the

modified electrode. Transmission electron microscopy (TEM,

Hitachi-7500. Japan) and Field emission scanning electron microscopy

(FESEM, JSM-7800F. Japan) were employed to characterize the surface

area and pedal-like structure of Pt@AgNFs and the size of AuNPs.

Energy dispersive spectrometer (EDS, Oxford X-MaxN. Britain) was

used to analyze the elements of the Pt@AgNFs. Scanning electron

microscopy (SEM, HITACHI, S-3000 N. Japan) was employed for AB

morphological analysis.

2.3. Preparation of AB and AuNPs

Firstly, 1.0 mg of AB was dispersed into 4.0 mL of 0.5 wt% chitosan

solution with the aid of ultrasonic agitation to obtain a homogeneous

suspension. And the solution was kept in an Eppendorf tube.

Gold nanoparticles (AuNPs, approximately 20 nm) were synthesized

by means of citrate reduction of HAuCl4 according to the literature (Yang

et al. 2016a) with a little modification. Briefly, 100 mL of 0.01% (w/v)

HAuCl4 was stirring and boiling for 15 min, and 2 mL of 1% trisodium

citrate was quickly added to above solution. Then, the mixture solution

was continued to stir another 15 min still the color changed from pale

yellow to wine red. Finally, after cooled to room temperature (RT), the

AuNPs solution was stored in brown flask at 4 °C for future use.

2.4. Synthesis of Pt@AgNFs

The innovative method for synthesizing the novel Pt@Ag

nanoflowers (Pt@AgNFs) is the key step to our research. Briefly, BSA (5

mg mL-1, 20 mL) and AgNO3 (10 mM, 10 mL) aqueous solutions were

mixed in a 100 mL beaker and the mixture was stirred at RT for 10 min,
AA (50 mg mL-1, 1 mL) was then added dropwise to the solution. The

color of the solution became light grey which indicated AgNFs was

formed successfully. Then H2PtCl6·6H2O (5.6 mM, 10 mL) was mixed

with the formed AgNFs solution. The mixture was stirred at RT for

another 10 min until the color became dark grey. It suggested that the

Pt@AgNFs were synthesized successfully. The resulting products were

collected and washed three times with water and ethanol, respectively.

The final precipitate was re-dispersed in deionized water for subsequent

use.

2.5. Synthesis of signal probe (Pt@AgNFs-Ab2)

Immobilization of capture antibody onto Pt@AgNFs was completed

according to the following steps (Scheme 1). Firstly, 1 mL Pt@AgNFs

were re-suspended with PBS, then 40 µL Ab2 (1 mg mL-1) was added into

Pt@AgNFs and the mixture was stirred well at 4 °C overnight. Finally,

the resulting signal probe (Pt@AgNFs-Ab2) was purified with three

circles of centrifugation and washing steps. Finally it was re-dispersed in

0.01 M PBS for future use.

2.6. Fabrication of proposed cytosensor

Prior to preparation procedure, gold electrode was firstly polished

with 0.3 and 0.05 μm alumina slurry. Then, a mirrorlike surface was
obtained. Followed by successive sonication with double distilled water

and ethanol, and then dried at RT. After the cleaning procedure, 10 μL of

AB solution was dropped on the bare electrode and dried at RT to obtain

AB modified gold electrode. Following, 10 μL of prepared AuNPs

solution was dropped on it and dried slowly in air. Protein G (40 µg mL-1)

was allowed to attach directly onto the gold surface by Au–NH2 chemical

bond for 3 h at 37 °C for efficient antigen–antibody binding.

Approximately 10 μL of Ab1 solution (100 μg mL-1) in 0.01 M PBS was

spread on the AuNPs/AB modified electrode and incubated at 37 °C, 1 h.

0.3% BSA solution was used to block the modified electrode overnight at

4 °C. The cytosensor was achieved after another round of washing with

0.01 M PBS stored at 4 °C until needed.

2.7. Electrochemical measurement of MCF-7 cells

Sandwich format was adopted. The resulting cytosensor was

incubated with different concentrations of MCF-7 cells for 40 min at

37 °C, and then immersed in the prepared Pt@AgNFs-Ab2 bioconjugate

(10 µL) solution for immunoreaction, followed by washing with 0.01 M

PBS to remove the unbound signal probe. The electrochemical

measurement was carried out with differential pulse voltammetry (DPV).

And the potential was ranged from 0 to 0.5 V in 0.01 M PBS.

2.8. Cell culture

MCF-7 and HeLa cells were cultured in Dulbecco's Modified Eagle

Medium (DMEM) supplemented with 10% fetal bovine serum (FBS,

Gibico), penicillin (100 mg mL-1) and streptomycin (100 mg mL-1) at

37 °C in a humidified atmosphere containing 5% CO2. According to

previously reported methods with a little modification (Mytych et al.

2015) the procedures can be generally summarized as follows: First of all,

all medium was removed and cells were washed with 0.01 M PBS.

Followed, the cells were digested with 0.25% trypsin/EDTA solution for

2–3 min. Then, the flesh medium was added to inhibit the activity of

trypsin. After centrifugation 500 rpm for 5 min, the cells were collected in

fresh medium. The medium was changed every 2–3 days.

The cells were separated from the medium by centrifugation at 500

rpm for 5 min and then washed twice with 0.01 M PBS. Followed, the

cell suspension was diluted to a certain concentration by 0.01 M PBS.

3. Results and discussion

3.1. Morphological characterization of AB and AuNPs

The SEM was used to characterize the AB and AuNPs. Fig. 1A

showed that there were plenty of particles in micrometre size distributed

uniformly, which indicated that AB could also provide the basis for the
subsequent stable signal. In Fig. 1B (the magnification of Fig. 1A), it

could be observed that the AB had a three dimensional crumpled and

wrinkled spherical structure with a uniform size of approximately 6 μm.

This particular morphology could increase the electrode effective surface.

As shown in Fig. 1C, the synthetic AuNPs were dispersed homogenously

with diameter size about 20 nm on average, which demonstrated that the

AuNPs was successfully synthesized.

3.2. Characterization of the Pt@AgNFs

The Pt@AgNFs played a crucial role in the cytosensor detection

performance. The structure and morphology of the Pt@AgNFs were

investigated using FESEM and TEM. Fig. 2A showed the overall

morphology of Pt@AgNFs. The product consisted of large quantities of

flower-like structures of average diameter 1 μm and they were well

dispersed in water. The enlarged FESEM image in Fig. 2B showed that

numerous thin petals assembled onto 3D flower-like structures. Fig. 2C

clearly revealed that Pt@AgNFs were composed of some small

nanoparticle aggregates which were covered by a dense of BSA proteins.

The pedal-like nanostructure could significantly increase the number of

self-assembled Pt and Ag nanoparticles. The TEM image of a single

nanoflower (Fig. 2D) displayed the typical example of nanoflower

structure, wrapped with a thin layer of BSA which confirmed the FESEM

results.

3.3. EDS analysis and biocompatibility of the Pt@AgNFs

EDS analysis was also performed to investigate the elemental

composition of the Pt@AgNFs, where Pt element and Ag element were

exclusively observed with high intensity (Fig. 3A). O and C peak was

also observed from BSA, indicating the successful synthesis of

Pt@AgNFs.

To evaluate the biocompatibility of the Pt@AgNFs, MCF-7 cells

were incubated with 50 μg mL-1 Pt@AgNFs for 0 h, 12 h and 24 h. Fig.

3B showed that when Pt@AgNFs were newly added, they distributed

dispersedly with the MCF-7 cells. After incubation for 12 h, as was

shown in Fig. 3C, it became attached to dish well. Fig. 3D revealed that

the morphology of MCF-7 cells was not changed after 24 h incubation

with 50 μg mL-1 Pt@AgNFs. This experiment indicated that the

Pt@AgNFs may be excellent biocompatible nanomaterials.

3.4. Electrochemical characterization of the proposed cytosensor

CV was regarded as a convenient and effective method for probing

the interface properties of surface-modified electrodes during the process

of fabrication. The electrochemical characterization of various modified

electrodes was conducted in K3[Fe(CN)6] solution (5 mM) containing 0.1

M KCl at a scan rate of 100 mV s-1. As shown in Fig. 4A, there was a

sharply increase current of 5 times over after successful modification of

AB and AuNPs (curve b) when compared with bare gold electrode (curve

a), which might be attributed to the enhancement of effective surface area

and high electrical conductivity of AuNPs/AB. As expected, the current

kept falling after the employment of the protein G (curve c), Ab 1 (curve

d), and BSA (curve e) blocking solution. This showed that the protein G,

Ab1, and BSA were successfully immobilized onto the electrode surface.

The current further decreased (curve f) when MCF-7 cells was captured

by its antibody, which was consistent with the fact that the hydrophobic

layer of the protein insulated the conductive support.

CV results were further confirmed via EIS characterization, which is

an effective method for studying the interface properties of surface

modified electrodes and the electron-transfer resistance at the electrode

surface. As depicted in Fig. 4B, curve a represents bare gold electrode,

and the resistance sharply decreased after the electrode was deposited

with AuNPs/AB (curve b). After the protein G was modified on the

electrode, the resistance increased due to their negative charge that block

the electron transfer (curved c). And increased further with the
modification of Ab1 (curve d), BSA (curve e), and MCF-7 cells (curve f),

this result was supported by the CV characterization.

3.5. Comparison of different states of the proposed cytosesnor

To demonstrate that AuNPs/AB can truly improve the signal

amplification, two types of experiments were carried out to investigate

the amplification properties. The DPV curves recorded in the two

situations were shown in Fig. S1. As was evident from curve b, the

change of electrochemical signal was much bigger than that of curve a.

The reason might be attributed to the enhancement of large specific

surface area and high electrical conductivity of the AuNPs/AB layer.

In order to prove that Pt@AgNFs can truly improve the signal

amplification, a cytosensor based on AgNFs was performed (Fig. S1B).

As expected, when detecting the same concentration of MCF-7 cells, the

peak current of Pt@AgNFs -based cytosensor (curve b) displayed bigger

than that of AgNFs-based cytosensor (curve a).

According to the above two comparison experiments, we hereby

confirm that AuNPs/AB, as large specific surface area nanomaterial, was

modified onto the gold electrode for sensitivity improvement. Meanwhile,

as the role of signal amplification strategy, Pt@AgNFs can also

remarkably improve the sensitivity of the cytosensor.

3.6. Optimization of experimental conditions

In order to obtain the best analytical performance of the cytosensor,

the conditions of the experiment including the concentration of protein G,

Ab2, the incubation time for MCF-7 cells–antibody reaction, the

concentration proportion of Pt@AgNFs and Ab2 have been optimized. In

these experiments, 106 cells mL-1 was used as an example. The

amperometric measurements were performed in the PBS (0.01 M, 50 mL)

containing 20 mM H2O2.

The stability of the proposed cytosensor was relied on the protein G

immobilized on surface of the electrode. So the concentration (10 µg

mL-1, 20 µg mL-1, 30 µg mL-1, 40 µg mL-1, 50 µg mL-1, and 60 µg mL-1)

was optimized by DPV respectively. The highest current was received

when 40 µg mL-1 of the concentration was used (Fig. S2A). Thus, the

optimum concentration of proG was considered as 40 µg mL-1.

The concentration of Ab1 was also a very important condition. The

amperometric response increased sharply with the increasing

concentration of Ab1, and reached a platform at 100 µg mL-1, then

decreased very slightly when the Ab1 concentration was over 100 µg mL-1

(Fig. S2B). These results demonstrated that the maximum peak current

could be obtained with 100 µg mL-1 Ab1.

The incubation time of MCF-7 cells and antibody also played a great
influence and it was studied in the range from 10 to 60 min. As shown in

Fig. S2C, the current response increased rapidly with the incubation time

increasing initially, but further increasing incubation time didn’t

significantly change the signal. Hence, 40 min was chosen as the optimal

MCF-7 cells and antibody incubation time.

Furthermore, the proportion of Pt@AgNFs and Ab2 was another

important parameter of the cytosensor. Higher or lower proportion of

Pt@AgNFs and Ab2 may lead to deterioration of the catalytic

performance for the reduction of H2O2. As seen in Fig. S2D, the current

response dramatically increased with the increasing proportion from

100:1 to 25:1, and then the current change decreased when the proportion

ranged from 25:1 to 10:1. The current variations declared that the

appropriate proportion of Pt@AgNFs and Ab2 not only ensured the

successfully specific binding between MCF-7 cells and antibody but also

efficiently enhanced the catalytic performance. Therefore, the proportion

of 25:1 had become the optimum proportion for this study.

3.7. Detection sensitivity and selectivity

On the basis of the optimal conditions, the prepared immunosensor

was applied for the MCF-7 cells detection. The as-prepared

electrochemical cytosensor was incubated with MCF-7 cells at different

concentrations. The dependence of the current response towards the

reduction of H2O2 on the concentration of MCF-7 cells was shown in Fig.

5A. When the concentration of MCF-7 cells increased, the current

response increased accordingly. As seen in the calibration plots of Fig. 5B,

there is a good linear relationship between the current response and the

logarithm of the concentrations of MCF-7 cells in the range from 20 to

1×106 cell mL-1. The linear regression equation was Y=14.796Log[C] +

5.671, with a linear correlation coefficient (R2) of 0.997, where Y was the

peak current of the cytosensor and C was the concentration of MCF-7

cells. The detection limit could be estimated as 3 cells mL-1 at a

signal-to-noise ratio of 3.

Moreover, the cytosensor showed a specific response against MCF-7

cells. HeLa cells (EpCAM-negative) were selected to evaluate the

specificity of this cytosensor at the same concentration under optimized

conditions. As Fig. 5C showed, compared with blank, no remarkable

change was observed toward 1×106 cell mL-1 of HeLa cells. While the

current response of MCF-7 cells with the same concentration was

relatively high. Moreover, the current response of a mixture solution

(1×106 cell mL-1 of MCF-7 and HeLa cells) was almost the same with

1×106 cell mL-1 of MCF-7 cell. These DPV responses indicated that the

proposed electrochemical cytosensor displayed a high specificity toward

MCF-7 cells.

Furthermore, the comparison of the performances of cytosensors

reported recently was shown in Table S1. It indicated that the reported

cytosensor exhibited lower detection limit and was a promising candidate

for quantitative recognition.

3.8. Stability and reproducibility

In this study, the stability of the immunosensor was investigated.

When the cytosensor was stored in refrigerator at 4 °C for 15 d, 90.46%

of its initial current response was retained compared with the freshly

prepared electrode, which suggested a satisfactory stability of the

established cytosensor. The result indicated that the cytosensor had an

acceptable stability which can be ascribed to the good stability of

substrate materials and the excellent biocompatibility of Pt@AgNFs.

The electrode-to-electrode reproducibility was also tested. Five

freshly prepared modified electrodes were used to detect the same

concentration (104 cells mL-1 and 105 cells mL-1) of MCF-7 cells. All five

electrodes exhibited almost similar current response, and the relative

standard deviation was 1.4% and 1.8%. The acceptable repeatability

demonstrated that if we conduct the assay as the proposed procedure, we

could obtain stable and coincident results.

3.9. Detection in blood samples

 To prove the performance of the fabricated cytosensor for detection

of CTCs in the blood samples, we detected the analytes in human blood

samples (provided by the first affiliated hospital of Chongqing Medical

University). Blood samples spiked with MCF-7 cells at concentrations of

1000, and 10,000 cells mL-1 were tested using the proposed cytosensors.

The recovery results agreed with the spiked amounts of MCF-7 cells and

the corresponding results are given in Table S2. The slight difference

between the measured and actual values could be due to the effect of the

blood samples, because they contained a small amount of analytes. These

experiments indicated that this strategy was a promising candidate for

quantitative recognition of MCF-7 cells, implying a good prospect in

clinical application.

4. Conclusions

In this research, an ultrasensitive sandwich-type cytosensor based on

AuNPs/AB and Pt@AgNFs was successfully fabricated and realized the

quantitative determination of CTCs. AuNPs/AB, as an ideal

biocompatible nanomaterial, was proved by this experiment for their

splendid electron transfer capability. Pt@AgNFs were utilizied as the

signal label, which significantly amplify the current signal. On the basis

of those merits mentioned above, the signal improvement was achieved

and the relatively low detection limit of 3 cells mL-1 (S/N=3) was
obtained, which was more sensitive compared with other methods.

Meanwhile, the novel fabricated cytosensor was also specific enough to

distinguish CTCs from the serum sample of patients. Therefore, the

developed cytosensor has provided a promising capacity to achieve the

precise detect for CTCs in clinical application.

Acknowledgments

This work was financially supported by National Natural Science

Foundation of China (No.81772255 to Wenli Feng, No.81672112 to

Guoming Xie, No.81500129 to Zhenglan Huang), Graduate Scientific

Research and Innovation Project of Chongqing (CYS16138), Chongqing

Yuzhong District Science and Technology Project (20140108), Scientific

and Technological Research Program of Chongqing Municipal Education

Commission（Grant No.KJ1500215), Top Talent Project of Chongqing

Medical University (BJRC201716), Key project of Education Department

of Sichuan（NO. 16ZA0181).

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Scheme 1. Schematic illustration of the electrochemical cytosensor

assembly process.

Fig. 1 SEM (A, B from lower magnification to higher magnification

images of AB) and TEM (C) of AuNPs.

Fig. 2 FESEM (A, B from lower magnification to higher magnification)

and TEM (C, D) images of Pt@AgNFs.

Fig. 3 (A) EDS pattern of Pt@AgNFs, (B) Morphology of MCF-7 cells

with newly added Pt@AgNFs (C) Morphology of MCF-7 cells incubated

with Pt@AgNFs for 12 h (D) Morphology of MCF-7 cells incubated with

Pt@AgNFs for 24 h.

Fig. 4 CVs (A) and EIS (B) spectra obtained from different electrodes: (a)

bare gold electrode, (b) AuNPs/AB/Au electrode, (c) Protein G/AuNPs/

AB/ Au electrode, (d) Ab1/Protein G/AuNPs/AB/Au electrode, (e) BSA/

Ab1/Protein G/AuNPs/AB/Au electrode, (f) MCF-7 cells/ BSA / Ab1/

Protein G/AuNPs/AB/Au electrode in phosphate buffer solution (pH 7.4)

containing 5 mM [Fe(CN)6]- 3/-4 and 0.1 M KCl (scan rate of CV: 0.1 V

s-1).

Fig. 5 (A) DPV responses of the cytosensor incubated with MCF-7 cells

of different concentrations in 0.01 M phosphate buffer (pH 7.5) :(a) 0, (b)

20, (c) 1×102, (d) 1×103, (e) 1×104, (f) 1×105 and (g) 1×106 cells mL-1 (B)

Calibration curve of the cytosensor for MCF-7 cells. (C) Specificity of

the proposed cytosensor. The RSD were 1.39%, 1.68%, 2.28%,

respectively. Error bars=RSD (n=5).

Highlights:

 AuNPs/Acetylene black showed strong electron–transfer ability and was firstly

employed in electrochemical cytosensor to remarkably enhance the sensitivity.

 The innovatively synthesized Pt@Ag nanoflowers were used to amplify

electrochemical signal for the very first time.

 Protein G was deposited on the electrode for the oriented immobilization of

capture antibody.

 The detection limit of this cytosensor could be estimated as 3 cells mL-1.