Magneto-Mediated Electrochemical Sensor for Simultaneous Analysis of Breast Cancer Exosomal Proteins

Article abstract of DOI:10.1021/acs.analchem.0c00106

Breast cancer is a heterogeneous disease, and it lacks special tumor markers. Exosomes, new noninvasive biomarkers, with the proteins on the exosome surface show potential for the diagnosis and prognosis of a tumor. However, assessing the variations of exosomal proteins still faces significant challenges. Herein, a magneto-mediated electrochemical sensor based on host-guest recognition has been developed for simultaneous analysis of breast cancer exosomal proteins. Magnetic beads (MB) modified with CD63 aptamer was first employed to capture exosomes. Silica nanoparticles (SiO2 NPs) was modified with MUC1, HER2, EpCAM, and CEA aptamers for specific exosomal proteins identification, respectively, and functionalized with N-(2-((2-aminoethyl)disulfanyl)ethyl) ferrocene carboxamide (FcNHSSNH2) as the signal molecule. The sandwich structure (MB-exosomes-SiO2 NPs probe) was separated by a magnet, and N-(2-mercaptoethyl) ferrocene carboxamide (FcNHSH) was released to the supernatant by the addition of reductants (dithiothreitol, DTT) that break the disulfide bond of FcNHSSNH2. FcNHSH and the graphene oxide-cucurbit [7](GO-CB[7]) modified screen-printed carbon electrode (SPCE) was employed to monitor the oxidation current signals. In this way, four tumor markers on different breast cancer cells (MCF-7, SK-BR-3, MDA-MB-231, and BT474) derived exosomes were sensitively detected. Furthermore, the present assay enabled accurate analysis of exosomes from breast cancer patients, suggesting the potential of exosome analysis in clinic diagnosis.

Full text of DOI:10.1021/acs.analchem.0c00106

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Article
Magneto-mediated electrochemical sensor for
simultaneous analysis of breast cancer exosomal proteins
Yu An, Rui Li, Fan Zhang, and Pingang He
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.0c00106 • Publication Date (Web): 11 Mar 2020
Downloaded from pubs.acs.org on March 12, 2020
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Magneto-mediated electrochemical sensor for simultaneous analysis of
breast cancer exosomal proteins
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Yu An, Rui Li, Fan Zhang*, Pingang He.
School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241,
P.R. China. Phone & Fax: +86-21-54340049; Email: fzhang@chem.ecnu.edu.cn
ABSTRACT: Breast cancer is a heterogeneous disease, and it lacks special tumor markers. Exosomes, a new noninvasive biomarker,
with the proteins on exosome surface show potential for the diagnosis and prognosis of tumor. However, assessing the variations of
exosomal proteins still faces significant challenges. Herein, a magneto-mediated electrochemical sensor based on host-guest
recognition has been developed for simultaneous analysis of breast cancer exosomal proteins. Magnetic beads (MB) modified with
CD63 aptamer was first employed to capture exosomes. Silica nanoparticles (SiO2 NPs) was modified with MUC1, HER2, EpCAM
and CEA aptamers for specific exosomal proteins identification, respectively, and functionalized with N-(2-((2-
aminoethyl)disulfanyl)ethyl) ferrocene carboxamide (FcNHSSNH2) as signal molecule. The sandwich structure (MB-exosomes-SiO2
NPs probe) was separated by a magnet and N-(2-mercaptoethyl) ferrocene carboxamide (FcNHSH) was released to supernatant by
the addition of reductants (dithiothreitol, DTT), that break the disulfide bond of FcNHSSNH2. FcNHSH and the graphene oxide-
cucurbit [7](GO-CB[7]) modified screen-printed carbon electrode (SPCE) was employed to monitor the oxidation current signals. In
this way, four tumor markers on different breast cancer cells (MCF-7, SH-BR-3, MDA-MB-231 and BT474) derived exosomes were
sensitively detected. Furthermore, the present assay enabled accurate analysis of exosomes from breast cancer patients, suggesting
the potential of exosome analysis in clinic diagnosis.
derived exosomes can be employed for the diagnosis of breast
cancer.16,17 Therefore, exosomes, as a new biomarker, can be
employed for the detection of breast cancers. In particular,
evaluation of exosomal surface proteins has important research
implication for the diagnosis and prognosis of tumor.18,19
However, analyzing the subtle variations of exosomal proteins
among different cell subtypes still faces significant challenges
due to lack of adequately accurate and sensitive assay platforms.
Introduction
Breast cancer is one of the common malignant tumors that
endangers women’s healthy and the incidence rate is increasing
year by year all over the world.1 It is a heterogeneous disease,
and there are various subtypes with different clinical behaviors.2
The protein expression level has important diagnostic value for
clinical diagnostic and classification due to the different
expression in different subtypes.3-5 Tumor markers are widely
used in the diagnosis and prognosis of tumors. However, there
is still a lack of specific tumor markers for breast cancer to
date.6,7 Hence, combined detection of tumor markers is of great
significance for early diagnosis, clinical detection and prognosis
of breast cancer.
Exosomes are lipid bilayer membrane vesicles (30-150 nm in
diameter) secreted by numerous cell types, and ubiquitous
presence in saliva, serum, urine, tears and other body fluids.8-10
Exosomes transport various molecular contents of the cell from
which they originate, including proteins and nucleic acids.11,12 In
particular, the exosomes carry a variety of tumor-specific
proteins on their surfaces, which can guide various pathological
and physiological processes in many signaling pathways.13-15
Early diagnosis and treatment of breast cancer are the key to
improve survival rate. Studies have shown that saliva and serum
Currently, there are various methods for the determination of
exosomal proteins. Flow cytometry is used for the high-
throughput detection of exosomes, but the vesicles with a
diameter <100 nm are easy to miss, which decreases the
accuracy of the measurement.20 Mass spectrometry, enzyme-
linked immunosorbent assays (ELISA) and western blot can
analyze the abundant exosomal proteins, but required a large
number of samples and complex procedures, limiting the
application in clinical research.21,22 Recently, fluorescence
23
24
sensing
and nanoplasmonic sensing were employed for
exosomes analysis with high sensitivity. However, the former
was susceptible to photobleaching and the latter required
expensive instrument. Additionally, electrochemical platform
was used for exosomal proteins profiling.25,26 Lee et al.
developed an eight-channel sensor for the simultaneous analysis
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of multiple exosome markers based on an integrated magneto-
(St. Louis, MO). Cyclohexane, 1-hexanol, triton X-100 and
electrochemical assay.26 However, it requires specialized
equipment. Therefore, there is an urgent need to detection
exosomal proteins in a convenient and reliable method.
silicon tetraacetate (TEOS) were purchased from the Sinopharm
Chemical Reagent Co. (Shanghai, China). Graphite oxide was
obtained from the Jcnano Chemical Industry Co., Ltd. (Nanjing,
China).
Dithiothreitol
(DTT),
and
In this work, an electrochemical sensor based on host-guest
recognition was developed for the simultaneous determination
of tumor exosomal proteins (Scheme 1). Mucin 1 (MUC1)
protein is a high molecular weight glycoprotein, which is highly
and abnormally expressed in breast cancer.27 Human epidermal
growth factor receptor-2 (HER2) overexpression can increase
the invasive and metastasis ability of tumor cancer, and is an
important predictor and prognostic marker for breast cancer.28
Epithelial cell adhesion molecule (EpCAM) is highly expressed
in almost all adenocarcinomas and affects the occurrence of
epithelial-mesenchymal transformation of breast cancer cells.29
Carcino-embryonic antigen (CEA) is highly specific and
sensitive for the detection of breast cancer, which is one of the
independent prognostic indicators of breast cancer.30 Hence,
MUC1, HER2, EpCAM and CEA proteins were employed for
combined detection of breast cancer in this work. CD63 is a
member of four transmembrane protein superfamily, and is
widely and highly expressed on the surface of many breast
cancer exosome types.31-33 Therefore, CD63 aptamers were
modified on the magnetic beads (MB) for tumor exosomes
capture. The silica nanoparticles (SiO2 NPs) were modified with
MUC1, HER2, EpCAM and CEA aptamers for specific
exosomal proteins identification, respectively. Then,
FcNHSSNH2, which has multiple functional groups, including
amino groups, disulfide bond and ferrocene, was combined with
SiO2 NPs by the reaction of the amino group and the aldehyde
group, and used as signal molecule. When exosomes were
present, the MB probe and SiO2 NPs probe formed the sandwich
structure, which was separated from unbound SiO2 NPs probes
by a magnet and FcNHSH was released to supernatant by the
addition of reductants (dithiothreitol, DTT) that break the
disulfide bond of FcNHSSNH2. Subsequently, the graphene
oxide - cucurbit[7] (GO-CB[7]) modified screen-printed carbon
electrode (SPCE) could form stable complexes with FcNHSH
through host-guest interaction, which effectively avoided the
modification of exosomes onto the electrode. Exosomal proteins
were further quantified by the oxidation current signal of
FcNHSH. In this way, four tumor markers on the surface of
exosomes from different breast cancers (MCF-7, SH-BR-3,
MDA-MB-231 and BT474) were accurately and sensitively
detected, which has potential application in early diagnosis,
clinical detection and prognosis of breast cancer.
trimethoxysilylpropyldiethylenetriamine
(DETA)
cucurbituril (CB[7]) were purchased from Sigma-Aldrich (St.
Louis, MO). Rabbit anti-human MUC1 polyclonal antibody,
rabbit anti-human HER2 polyclonal antibody, rabbit anti-human
EpCAM polyclonal antibody, rabbit anti-human CEA
polyclonal antibody and HRP conjugated rabbit polyclonal
secondary antibody were purchased from Abcam Pic.
(Cambridge, UK). The bicinchoninic acid (BCA) protein assay
kit and radio-immunoprecipitation assay (RIPA) lysis buffer
were obtained from the Beyotime Biotechnology Co. Ltd.
(Shanghai, China). Dulbecco’s modified eagle medium
(DMEM), roswell park memorial institute (RPMI) 1640
medium, fetal bovine serum (FBS), trypsin/EDTA solution,
penicillin/streptomycin (P/S) and dulbecco’s phosphate
buffered saline (PBS) were obtained from Thermo Fisher
Scientific Inc. (Waltham, MA, USA). Exosome-depleted fetal
bovine serum and the ExoQuickExosome Isolation Kit were
purchased from System Biosciences Inc. (SBI, USA). The
human breast cancer cell line (MCF-7, SK-BR-3, MDA-MB-
231 and BT474) were obtained from the Chinese Academy of
Sciences (Shanghai, China). The filters (0.22 μm in aperture)
were purchased from the Millipore Corp. (Bedford, MA, USA).
A Tris-HCl buffer (5 mM, pH = 7.5) containing 0.5 mM
EDTA and 1 M NaCl was used as the washing buffer. A
phosphate buffered saline (PBS, 10 mM, pH = 7.4) containing
0.1 M KCl was employed as the electrochemical detection
solution. All solutions were prepared with deionized water (DI,
≥ 18.2 MΩ∙cm-1) by a Milli-Q water purification system
(Millipore Corp., Bedford, MA, USA).
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Instrumentation. MCF-7, SK-BR-3, MDA-MB-231 and
BT474 cells were cultured in a humidified incubator (NuAire,
USA). The transmission electron microscopy (TEM)
characterization of exosomes and SiO2 NPs were performed on
a Hitachi-7700 instrument (Tokyo, Japan) and JEOL JEM-2100
(Hitachi, Japan), respectively. Zeta potential characterization
1
were performed on a ZetasizerNano (Malvern, England). H
NMR and 13C NMR were performed using AVANCEⅢ 500
(Bruker, Germany). Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) was performed on a BioRad
electrophoresis apparatus and imaged on a Typhoon 9410
variable mode imager (Amersham, USA). Electrochemical
measurements were carried out on a CHI1030C electrochemical
workstation (Chenhua, Shanghai, China).
Experimental Section
Reagents and Materials. All oligonucleotides were
synthesized by the Sangon Biological Engineering Technology
& Services Co., Ltd. (Shanghai, China), and listed in Table S1.
Preparation of FcNHSSNH2. Cystaminedihydrochloride
(2.55 g, 11.33 mM) was first suspended in 50 mL of CHCl3, and
then treated with an aqueous solution of NaOH (2 M, 50 mL).
The aqueous phase was extracted three times with CHCl3. The
combined organic phase was dried overnight with anhydrous
Ferrocenecarboxylic
acid,
N-hydroxysuccinimide
and
cystaminedihydrochloride were purchased from Sigma-Aldrich
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MgSO4. The solvent was removed by rotary evaporation to yield
quantification of the exosomal protein concentration. The
cystamine.34 Ferrocene carboxylic acid (1.03 g, 4.5 mM) was
dissolved in 70 mL of anhydrous CH2Cl2. Then, N-
hydroxysuccinimide (0.61 mg, 5.3 mM) and N-(3-
protein samples (10 μg) was subjected to 10% SDS-PAGE, and
further electrotransferred to a nitrocellulose filter membrane.10
Afterwards, the membranes were blocked with 5% BSA and
incubated overnight at 4 ºC with the following monoclonal
antibodies: α-MUC1, α-HER2, α-EpCAM and α-CEA. Next, the
blots were incubated for 1 h with HRP-conjugated rabbit
polyclonal antibodies and the protein strips was imaged by a Gel
Image System.
Preparation of Magnetic Beads (MB) Probe. The
preparation of aptamers-modified magnetic beads was carried
out as reported previously.39 Briefly, 12.5 μL of streptavidin-
modified magnetic beads (10 mg/mL) were first washed three
times with 5 mM Tris-HCl buffer (pH 7.5) containing 0.5 mM
EDTA and 1 M NaCl, and resuspended in 50 μL of 0.1 M PBS
(pH 7.4) containing 0.1M KCl. Then, 3 μL of 100 μM biotin-
CD63 aptamer was added to the above solution with gentle
shaking at 37 ºC for 2h, followed by washing three times with
0.1 M PBS. Next, the pellets were resuspended in 50 μL of 0.01
M PBS.
Preparation of SiO2 NPs Probes. Firstly, the SiO2
nanoparticles (SiO2 NPs) were prepared as the previous work.40
Briefly, 30 mL of cyclohexane, 7.2 mL of n-hexanol and 7.2 mL
of Triton X-100 were stirred for 5 min. Then, 1.2 mL of
ultrapure water was added to the solution, and the mixture was
stirred for 30 min at room temperature. Subsequently, 400 μL of
TEOS and 240 μL of NH3·H2O (25 wt%) were slowly dripped
into the mixture and vigorously stirred for 24 h at room
temperature. The defined amount of ethanol was added to the
reaction mixture to break up the microemulsion, and the solution
was centrifuged and washed three times with ethanol and water,
respectively. The SiO2 NPs were finally resuspended in 10 mL
of ultrapure water and stored at 4 ºC for further use. Afterwards,
100 μL of SiO2 NPs were aminated in 5 mL of aqueous solution
containing 5% DETA and 1 mM HAc with shaking at 37 ºC for
1 h.41 The solution was centrifuged and washed three times with
water. After that, the SiO2 NPs pellets were added to 5 mL of
5% glutaraldehyde for 1 h with gentle shaking at 37 ºC.
Subsequently, the solution was centrifuged and washed three
times with water, and then was resuspended in 100 μL of 0.1 M
PBS buffer. 5 μL of 100 μM amino-modified aptamer was added
into the SiO2 NPs solution with shaking for 2 h at 37 ºC. Next,
1 μL of 100 mM FcNHSSNH2 was added into the mixture and
allowed to react for 2 h at 37 ºC with gently shaking. The
mixture was then washed three times with 0.1 M PBS and
resuspended in 50 μL of 0.01 M PBS buffer.
dimethylaminopropyl)-N’-ethycarbodiimide
hydrochloride
(0.98 mg, 5.2 mM) were added, and the reaction mixture was
stirred overnight at room temperature. After the solution was
washed three times with water, the combined water phases were
extracted with CH2Cl2 and the combined organic phases were
dried overnight with anhydrous MgSO4. The solvent was
removed in vacuo, and the reaction mixture was purified by
silica gel chromatography (diethyl ether) to yield ferrocene-
carboxylic acid N-succinimide ester (FcNHS).35 The obtained
FcNHS (1.56 g, 4.77 mM) was then dissolved in 50 mL of
CH2Cl2, followed by the treatment of cystamine (1.21 g, 7.96
mM) and Et3N (2 mL) under stirring for 12 h at room
temperature. After the reaction mixture was washed three times
with water, the organic phases were dried over MgSO4. The
solvent was removed in vacuo, and the reaction mixture was
purified by silica gel chromatography (CH2Cl2: MeOH = 9:1) to
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give
N-(2-((2-aminoethyl)disulfanyl)ethyl)
ferrocene
carboxamide (FcNHSSNH2) as a yellow solid.34
Cell Culture and Exosome Isolation. The MCF-7 cells and
MDA-MB-231 cells were cultured in DMEM medium
containing 10% FBS and 1% P/S in a humidified incubator of
5% CO2 and 95% air at 37 ºC. The SK-BR-3 cells and BT474
cells were cultured in 1640 medium containing 10% FBS and
1% P/S. All cells reaching 80% confluence were detached with
0.25% trypsin/EDTA and centrifugated (800 rpm, 5 min) to
allow for subculturing. Exosomes were isolated as reported
previously.36 In brief, the exosome-depleted FBS medium from
1×106 cells were collected after 48 h. Then, the medium was
centrifuged successively at 300 g for 10 min to eliminate
apoptotic cells, and 2000 g for 20 min and 10000 g for 30 min
at 4 ºC to remove the cell debris. Finally, it was filtered through
a 0.22 μm filter. Afterwards, the supernatant was further
centrifuged at 100000 g for 1 h at 4 ºC to collect the exosomes,
followed by washing with PBS and another ultracentrifugation.
Finally, the exosomes were resuspended in 100 μL of 0.01 M
PBS and stored at -80 ºC.
Characterization of Exosomes. The TEM characterization
of exosomes from MCF-7 cells were carried out according to
previous protocol.10,37 Briefly, 5 μL of exosomes in PBS were
dropped on the carbon-coated copper grid for 20 min, and then
the remaining solution was evaporated in a dry environment.
The pellets were further carried out via negative staining using
1% phosphotungstic acid for 10 s. The remaining solution was
absorbed by filter paper and imaged using TEM.
Electrochemical Detection. The SPCE (3 mm in diameter)
with four channels was first washed with water. Then, 7 μL of
uniform GO-CB[7] suspension droplets was dropped to the
SPCE and dried at room temperature.42 The procedures for
exosomes detection was as follows. First, 50 μL of MB probe
was incubated with exosomes under gentle shaking at 37 ℃ for
3 h, followed by washing with 0.01 M PBS. Then, 50 μL of SiO2
Western blot was employed for the analysis of exosomal
proteins
according
to
previously
described
with
modification.10,38 The exosomes of MCF-7 cells, SK-BR-3 cells,
MDA-MB-231 cells and BT474 cells were lysed with RIPA
buffer, respectively. Then, the BCA method was used for the
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NPs probes were added to the MB probe. After incubation for 3
Feasibility of this Strategy. The morphology of MCF-7
h with gentle shaking, the complexes were washed with 0.01 M
PBS three times. Next, 50 μL of 10 mM DTT solution was added
and incubated with the above complexes for 30 min to break the
disulfide bond.43 Afterwards, 7 μL of the supernatant solution
was dropped on the GO-CB[7] modified SPCE for host-guest
recognition at 37 ℃. The modified SPCE was rinsed and DPV
measurements were determined in 0.1 M PBS. In the three-
electrode system, SPCE, Ag/AgCl electrode and platinum wire
were employed as the working electrode, the reference electrode
and the auxiliary electrode, respectively. The DPV signals were
detected from 0.2 V to 0.8 V with the pulse amplitude of 50 mV,
pulse width of 50 ms and pulse period of 0.2 s.
Detection of Exosomes in Human Serum. Shanghai Ruijin
Hospital provided us with serum samples from breast cancer
patients and healthy individuals. Exosomes from serum samples
were isolated by ExoQuickExosome Isolation Kit according to
previous protocol.10 The human serum was centrifuged
successively at 3000 g for 15 min, which could eliminate the cell
debris. The supernatant was further collected in a new tube and
the isolation reagent was added at the ratio of 1:4. The samples
were incubated for 1 h at 4 ℃ and centrifugated at 1500 g for 30
min to obtain exosome pellets. Then, the exosome pellets were
resuspended in 500 μL of 0.01 M PBS solution and stored at -80
ºC. The electrochemical measurement procedures were
consistent with those for exosomes detection described above.
exosomes was observed on the TEM image. As shown in Figure
1A, these exosomes presented a typical cup shape morphology.
Meanwhile, these exosomes contained double-walled lipid
membrane layers at a diameter of 50-100 nm, consistent with
reported exosomes.10,37 Meanwhile, nanoparticle tracking
analysis (NTA) was used to characterize the concentration of all
isolated exosomes. To confirm the modification of SiO2 NPs
probe, TEM and Zeta potential were employed. As shown in
Figure 1B, SiO2 NPs have a smooth spherical structure with a
diameter of ~100 nm, as well as good dispersion. Figure 1C
exhibits the surface charge change with the ammoniation of SiO2
NPs by DETA (zeta potential, -46.3 mV) to obtain SiO2 NPs-
NH2 (zeta potential, 37.5 mV). After the surface modification of
glutaraldehyde, the zeta potential decreases to 29.5 mV,
indicating the successful functionalization of aldehyde group,
obtaining SiO2 NPs-CHO.44,45 When the aptamers with negative
charges were coupled to the nanoparticles (SiO2 NPs-Apt), the
zeta potential was dramatically reduced to -39.0 mV. While, the
zeta potential is increased to -3.36 mV, when FcNHSSNH2
molecule was modified on the nanoparticles to generate SiO2
NPs-FcNHSSNH2. These results prove the successful surface
modification of SiO2 NPs probe. Using MUC1 marker on MCF-
7 exosomes as the model, the DPV responses were determined
with and without the exosomes and exhibited in Figure 1D.
Clearly, when the exosomes were present in the system at the
concentration of 1.2×106 particles/μL, the current signal with
exosomes was about 7 times that without exosomes. These
results show that the magneto-mediated electrochemical sensor
could be used for the detection of exosome markers.
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1
Figure S1 displayed the H NMR and 13C NMR of FcNHS
and FcNHSSNH2, respectively. 1H NMR of FcNHS (500 M Hz,
CDCl3) δ/ppm 4.96 (t, J=1.9 Hz, 2 H), 4.73 (t, J=2.0 Hz, 2 H),
4.43 (s, 5 H), 2.99-2.74 (m, 4 H); 13C NMR of FcNHSSNH2 (126
M Hz, CDCl3) δ/ppm 169.57, 167.40, 72.83, 70.79, 70.74, 64.27,
25.67. 1H NMR of FcNHSSNH2 (500 M Hz, DMSO-d6) δ/ppm
7.98 (s, 1 H), 4.78 (t, J=1.9 Hz, 2 H), 4.35 (t, J=1.9 Hz, 2 H),
4.18 (s, 5 H), 3.51-3.42 (m, 2 H), 2.89 (t, J=6.9 Hz, 2 H), 2.84
(dd, J=7.2 Hz, 4.9 Hz, 2 H), 2.78 (dd, J=7.2 Hz, 4.9Hz, 2 H); 13C
NMR of FcNHSSNH2 (126 M Hz, DMSO-d6) δ/ppm 169.58,
76.86, 70.41, 69.85, 68.61, 42.00, 41.27, 38.77, 37.92.
Scheme 1. Schematic representation of the magneto-mediated
electrochemical sensor for exosomal proteins analysis based on
host-guest recognition.
Results and Discussion
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Figure 2. Effects of (A) the ratio between MUC1 aptamer and
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Figure 1. (A) TEM image of the MCF-7 exosomes; (B) TEM
FcNHSSNH2 in MB probe, (B) the incubation time of MB probe
with exosomes, (C) the incubation time of SiO2 NPs probe with
exosomes, and (D) the recognition time of FcNHSH by GO-CB[7]
with 1.12×106 particles/μL MCF-7 exosomes. Each data point is the
mean of three measurements and the relative standard deviation
(RSD) is less than 6.3%.
image of SiO2 NPs; (C) Zeta potential of the SiO2 NPs probe;
(D) DPV signals with (a) and without (b) the MCF-7 exosomes
at concentration of 1.2×106 particles/μL.
Optimization of Detection Conditions. Taking the
determination of MUC1 marker on MCF-7 exosomes as the
model, the main experimental conditions were optimized to
improve the sensitivity of the detection. Figure 2A shows the
effect of the ratio between MUC1 aptamer and FcNHSSNH2 on
the electrochemical response. It could be observed that the
current signal reaches a maximum when the ratio of MUC1
aptamer and FcNHSSNH2 was 1:200. However, by increasing
the ratio from 1:200 to 1:100, the current response gradually
decreases, probably due to the reduction FcNHSSNH2 on SiO2
NPs. Thus, the optimal ratio was 1:200. Figure 2B exhibits that
DPV responses with increasing the incubation time of exosomes
with MB probe from 0.5 h to 4 h. Clearly, beyond 3 h, the current
signal remains almost constant. Hence, the optimal incubation
time of MB probe and exosome was 3 h. The reaction time of
SiO2 NPs probe with exosomes is another key factor for the
amount of MUC1 aptamer combined with exosomes. As shown
in Figure 2C, it could be observed that the current response first
increases as the incubation time increases from 1 h to 3 h and
then remains nearly constant in the range of 3 h -5 h. Thus, 3 h
was selected as the optimal incubating time of SiO2 NPs probe
with exosomes. Figure 2D illustrates the influence of the
incubation time of FcNHSH with GO-CB[7] modified SPCE.
The reduction peak current continuously increases in the range
from 2.0 h to 3.5 h. However, the current signal no longer
changed with further extension of incubation time, suggesting
that the host-guest recognition of FcNHSH molecular with GO-
CB[7] reaches saturation at 3.5 h.
Profiling of Protein Markers in Breast Cancer Cells-
derived Exosomes. Under the optimal detection conditions, the
subtle changes of MUC1 marker level in different breast cancer
cells-derived exosomes were determined at various
concentrations. From Figure 3A, it could be observed that there
is a linear relationship between current and the logarithm of
MCF-7 cells-derived exosomes concentration in the range of
1.2×103 - 1.2×107 particles/μL. The equation is as follows: ΔI =
0.3561 × lgc - 0.3305 (R2 = 0.9911), where ΔI is the difference
of current signal with and without the exosomes, and c
represents the concentration of exosomes. Figure 3B presents
the linear relationship between current and the logarithm of the
SK-BR-3 cells-derived exosomes concentration with the
equation of ΔI = 0.1022 × lgc+ 0.3624 (R2 = 0.9674), The
logarithm of the MDA-MB-231 cells-derived exosomes
concentration in the same range and the current response have
the following linear relationship: ΔI = 0.2232 × lgc- 0.0007 (R2
= 0.9920). For BT474 cells, the logarithm of exosome
concentrations and the current signals satisfy a linear
relationship with the equation of ΔI = 0.3295 × lgc - 0.2733 (R2
= 0.9938, Figure 3D). Clearly, the response slopes are
significantly different for these four kinds of breast cancer cells-
derived exosomes, suggesting the different expression level of
MUC1 on exosomes: MCF-7 cells-derived exosomes present
the most abundant MUC1, then BT474 cells-derived exosomes,
MDA-MB-231 cells-derived exosomes, and finally SK-BR-3
cells-derived exosomes. Thus, this developed sensor could be
able to distinguish the subtle changes of MUC1 level in different
exosomes and might allow identification of other tumor
markers.
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Figure 4. DPV responses of the magneto-mediated electrochemical
Figure 3. Analysis of MUC1 marker on (A) MCF-7, (B) SK-BR-3,
(C) MDA-MB-231 and (D) BT474 cells-derived exosomes at
different concentrations (1.2×103, 6.6×103, 1.2×104, 6.6×104,
1.2×105, 6.6×105, 1.2×106 and 1.2×107 particles/μL). Each data
point is the mean of three measurements and the relative standard
deviation (RSD) is less than 6.3%.
sensor for MUC1, HER2, EpCAM and CEA markers form the (A)
MCF-7, (B) SK-BR-3, (C) MDA-MB-231 and (D) BT474 cells-
derived exosomes at a concentration of 1.2×106 particles/μL.
The DPV current responses of various tumor markers were
summarized and presented as a heat map in Figure 5A.
Meanwhile, western blot (WB) as a traditional protein detection
method was employed for comparison (Figure 5B). Clearly, the
high expression of MUC1 protein is presented on MCF-7 and
BT474 exosomes, compared with the moderate level on MDA-
MB-231 exosomes and the minimum level on SK-BR-3
exosomes. HER2 protein is highly expressed on BT474
exosomes, followed by SK-BR-3, MDA-MB-231 and MCF-7
exosomes. For EpCAM protein, its expression level on MCF-7,
SK-BR-3, MDA-MB-231 and BT474 exosomes is successively
increased. CEA protein is expressed at a relatively low level
except on BT474 exosomes. The results indicate that the
expression levels of the tumor markers were significantly
different and related to the breast cancer cells-derived exosomes
types. The obtained protein information was basically consistent
with the result of WB. While, our method required less
exosomes sample than WB and had a higher sensitivity. Hence,
the sensor can be used to classify different subtypes of breast
cancer cells and allows non-invasive early diagnosis of breast
cancer by combined detection of tumor markers. Moreover,
compared with the reported methods (Table S2), our method
presents a comparable sensitivity and wider detection linear
range. More importantly, it has achieved a combined detection
of exosomal proteins for breast cancer, which could improve the
accuracy of detection, and thus shows a high potential in early
diagnosis, clinical detection and prognosis of breast cancer.
Furthermore, the expression of four tumor markers on these
breast cancer cells-derived exosomes were profiled. Figure 4A
presents the difference in the expression of MUC1, HER2,
EpCAM and CEA proteins on MCF-7 exosomes at a
concentration of 1.2×106 particles/μL. Clearly, MUC1 protein is
highly expressed, followed successively by EpCAM, HER2 and
CEA. The same order of protein expression appears on MDA-
MB-231 exosomes, while with different quantities (Figure 4C).
For SK-BR-3 exosomes (Figure 4B) and BT474 exosomes
(Figure 4D), the amount of EpCAM, HER2, MUC1 and CEA
proteins they carry both decrease successively, but the
determined values are obviously different. These results suggest
that this electrochemical sensor can be employed to the
distinguish the subtle changes of various tumor markers on
different exosomes.
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Figure 5. (A) Electrochemical responses represented as a heat
map highlighting the difference of four tumor markers on four
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types of breast cancer cells-derived exosomes; (B)
Corresponding results of Western blot analysis.
Figure 6. Detection of exosomal tumor markers in human serum
with breast cancer (patient, P) and non-cancer (healthy, H). Each
data point is the mean of three measurements and the relative
standard deviation (RSD) is less than 6.3%.
Reproducibility and Stability of the Sensor. Taking the
determination of MUC1 marker on MCF-7 cells-derived
exosomes as the model, the reproducibility and stability of the
electrochemical sensors were evaluated. Nine electrodes
modified under the same conditions were measured and the
relative standard deviation (RSD) of current for 1.2×106
particles/μL exosomes was found as 6.23%, reflecting a good
reproducibility of the electrochemical sensor (Figure S2A). To
investigate the stability, the modified electrodes were stored at
4 °C and measured every 7 days (Figure S2B). The sensor
retained almost 97.7% of the initial signal for 7 days. 14 days
later, the current decreased to 95.9%. It had 94.8% and 90.8%
of the initial values for 21 days and 28 days, respectively,
presenting an excellent stability of the sensor.
Conclusions
In summary, a magneto-mediated electrochemical sensor has
been developed to profile protein markers information in breast
cancer cells-derived exosomes. It employed the aptamers for the
specific recognition of four exosomal proteins and took
advantage of the SPCE for the simultaneous detection based on
host-guest recognition between GO-CB[7] and FcNHSH. This
sensor could differentiate the subtle variations of exosomal
proteins among different breast cell subtypes. Meanwhile, the
developed biosensor displayed great potential in the
determination of exosomes in serum from breast cancer patients
with high accuracy and easy operation, which is promising for
clinical diagnosis.
Determination of Exosomes in Serum from Breast Cancer
Patients. To demonstrate the practical ability of this sensor, the
exosomes at a concentration of 1.0×107 particles/μL derived
from human serum samples with breast cancer (patient, P) and
non-cancer (healthy, H) were analyzed. By comparing the
current responses, it could be found that the expression levels of
MUCI, HER2, EpCAM and CEA proteins on breast cancer
patient-derived exosomes were all higher than those on healthy
individual-derived exosomes (Figure 6). The results clearly
suggested that our method is suitable for analyzing the exosomes
in clinical samples.
Supporting Information
Additional information as noted in text. This material is
available
free
of
charge
via
the
Internet
at
http://pubs.acs.org.
Sequences of the oligonucleotides; NMR characterization of
FcNHS and FcNHSSNH2; Reproducibility and stability.
AUTHOR INFORMATION
Corresponding Author
*Fan Zhang, Phone
& Fax: +86-21-54340049, Email:
fzhang@chem.ecnu.edu.cn.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
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Vesicles. 2017, 6(1), 1340745.
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 21575042).
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