Article

Cite This: ACS Sens. 2019, 4, 1433−1441 pubs.acs.org/acssensors

Ultrasensitive and Reversible Nanoplatform of Urinary Exosomes for

Prostate Cancer Diagnosis
Ping Li,† Xiyuan Yu,† Wujuan Han,† Ying Kong,‡ Weiyang Bao,† Jiaqi Zhang,† Wancun Zhang,†
and Yueqing Gu*,†,‡
†
Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, No. 639 Longmian Avenue,
Jiangning District, Nanjing 211198, China
‡
State Key Laboratory of Natural Medicine, China Pharmaceutical University, No. 639 Longmian Avenue, Jiangning District,
Nanjing 211198, China
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*
S Supporting Information
Downloaded via CARLETON UNIV on July 17, 2020 at 12:10:09 (UTC).

ABSTRACT: Prostate cancer cell-derived exosomes in urine have been extensively studied recently and regarded as novel
biomarkers for cancer diagnosis and prognosis, which presents wide prospects in clinical applications. Sensitive detection and
specific capture methods are essential for exosomes analysis. Herein, a dual functional platform composed of superparamagnetic
conjunctions and molecular beacons (SMC-MB) is reported. The SMC-MB platform is designed based on aptamer
immunoaffinity with ultrasensitive detection efficiency and reversible isolation capacity, which, respectively, profit from
nonenzymatic amplification methods and magnetic separation along with restriction cleavage. It is noteworthy that exosomes
quantification was exactly amplified and transformed into single strand DNA detection. Correlated measurements evidence that
the limit of detection of SMC-MB is as low as ∼100 particles/μL in urine, and a linear relationship meets between the
logarithmic concentration of exosomes and fluorescence intensity of the molecular beacon. Furthermore, employing prostate
specific membrane antigen (PSMA) aptamer, the platform adapted to detect and capture PMSA-positive exosomes from urine
samples provides excellent diagnostic efficiency for prostate cancer (PCa). The expression of typical biomarkers of PCa, i.e.,
PSA and PCA3 mRNA, is significantly higher in PSMA-positive exosomes. Altogether, the platform and strategy described in
this paper are promising in urinary exosomes analysis and prostate cancer detection.
KEYWORDS: urinary exosomes, cancer biomarker, prostate cancer diagnosis, prostate specific membrane antigen, aptamer

E xosomes are nanoscale extracellular vesicles generated by

inward budding of intercellular endosomes which have
diameters of 30−150 nm, and have gained much attention due
to their abundance of bioinformation alongside the important
roles in intracellular communication.1−3 Cancer-derived
exosomes have been identified as important in cancer
development, progression, as well as drug resistance. Cancer
related pathogenic components, including mRNA (mRNA),
microRNA (miRNA), noncoding RNA (ncRNA), DNA, and
membrane and cytosolic proteins and lipids4−6 can be
delivered and exchanged through the double lipid membrane
vesicles, thus contributing to the recruitment and reprogram-
ming of the tumor microenvironment. 7,8 Accordingly,
exosomes are of great significance in cancer diagnosis, as the
amount of secreted exosomes together with cancerous nucleic
acids and proteins they contain allow the prediction of cancer
Received: April 1, 2019
Accepted: April 24, 2019
Published: April 24, 2019

© 2019 American Chemical Society 1433 DOI: 10.1021/acssensors.9b00621

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Figure 1. Schematic illustration of SMC-MB platform. (a) Construction of SMC-MB platform. (b) Procedure of SMC-MB platform in exosomes
analysis. (c) Principle of nonenzymatic amplification cycle. (d) Exosomes purification by restriction enzyme.
risk.9,10 Circulating exosomes in urine, blood, and saliva can be
noninvasive or minimally invasive biomarkers.11,12 Compared
with serum or plasma, urine yields large volumes and permits
routine collection.13 Urine exosomes have been proposed as
treasure chests of biomarkers especially for some tumors
located anatomically proximate to the urethra.14 Notably,
recent studies have proved that prostate cancer (PCa) derived
exosomes are enriched in urine after a slight prostate massage,
which assists the discovery and detection of prostate cancer.15
Even though prostate specific antigen (PSA) has shown
reasonable sensitivity for prostate cancer screening, diagnosis,
as well as prognosis, one of the drawbacks is low specificity,
such that benign hyperplasic conditions can also lead to PSA
increment.16 Thus, tumor-derived exosomes detection is a
novel approach to find predictive markers for PCa according to
numerous studies. Relatively, prostate specific membrane
antigen (PSMA) positive exosomes demonstrate obvious
merits in PCa diagnosis, and offer essential biomarkers for
the development of biosensor-based PCa detection methods.17
Although increasing studies have focused on urinary
exosomes, it is still challenging to isolate and detect exosomes
from the complex matrix. To date, conventional methods for
exosomes detection include transmission electron microscopy
(TEM), nanoparticle tracking analysis (NTA), Western blot,
and ELISA which are either time-consuming, unwieldy,
inconvenient, or expensive.18,19 The main techniques for
highly efficient separation of exosomes are ultracentrifugation,
immunoaffinity capture, exosome precipitation and micro-
fluidics-based methods.20−22 In addition, ultracentrifugation
serves as the gold standard for total exosomes isolation, while
immunoaffinity method can accurately capture exosomes with
specific surface proteins, but both of them are expensive,
irreversible and monofunctional in clinical separation.23
Recently, convenient and quick exosomes analysis methods
with high sensitivity and specificity have been reported.
Researchers prefer to isolate exosomes by magnetic separation
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along with electrochemical24,25 and microfluidic methods,26
surface enhanced Raman scattering (SERS)27 probes, and
fluorescent probes28 to detect exosomes simultaneously.
However, the limitation of low concentration as well as the
complex background of urine are cannot be ignored in
exosomes collection and detection. As a result, signal
amplification and reversible strategy should be further
developed.
Herein, a dual functional platform composed of a super-
paramagnetic conjunction and molecular beacon (SMC-MB)
was reported to separate and quantify PCa-related exosomes,
and convert exosome detection to single strand DNA (ssDNA)
detection. Typically, PSMA (prostate specific membrane
antigen) aptamer, a recognized PSMA target ligand with
high affinity and low immunogenicity was applied to capture
PSMA-positive exosomes.29 And ssDNAs complementary with
aptamers were divided into two parts in order to decrease the
hybridization energy with aptamers, and further increase the
competition capacity of exosomes to replace ssDNAs from
aptamers, as well as double the exosomes signal (single
exosome produces two ssDNA signal) which could be helpful
when exosomes concentration was low. According to previous
studies,30 two hairpin DNA probes (HP1, HP2) were designed
to initiate amplification cycle and detection of released ssDNAs
with low concentration. Particularly, HP2 was designed as a
molecular beacon, an “on−off” fluorescent probe which has
been widely used in various biomarkers detections.31,32 The
high sensitivity and efficiency of this platform were verified in
both cell medium and urine samples. In addition, different
from other irreversible immunoaffinity isolation methods,
restriction sites were introduced to purify target exosomes
from the superparamagnetic platform. Finally, exosomes were
further studied for cancerous properties. Above all, this
platform can be used to isolate and detect PCa related
exosomes, revealing application potential in prostate cancer
diagnosis and prognosis.
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Figure 2. Characterization of SMC-MB. (a) FTIR spectrum of Fe3O4 cores and conjunctions. (b) Absorbance intensity in supernatant before and
after aptamers reaction with Fe3O4 cores. (c) PAGE gel of different samples: Lane 1, standard DNA markers; lane 2, 0.5 mM ssDNA1; lane 3, 0.5
mM ssDNA2; lane 4, supernatant after ssDNA1 incubation; lane 5, supernatant after ssDNA2 incubation. (d) Absorbance intensity in supernatant
before and after ssDNA incubation. (e) Zeta potential of superparamagnetic Fe3O4 cores, Fe3O4-EDC, and Fe3O4 cores-aptamer and SMCs. (f)
Hydrodynamic diameters of superparamagnetic Fe3O4 cores and SMCs. (g) TEM image of superparamagnetic Fe3O4 cores. (h) TEM image of
superparamagnetic conjunctions (SMCs).

■ RESULTS AND DISCUSSION

Principle of SMC-MB Platform. The capture and
detection mechanism of the SMC-MB platform for PCa
related exosomes is exhibited in Figure 1. SMCs were
composed of superparamagnetic Fe3O4 cores and specific
aptamer-ssDNA complex (Figure 1a). All sequences applied in
this paper are listed in Supporting Information Table S1. After
incubation with exosomes samples, two steps were carried out
to quantify and purify exosomes, respectively. Due to higher
affinity between exosomes and aptamers, ssDNAs were
replaced and disassociated into supernatant and subsequently
separated from SMCs by magnet (Figure 1b). In such a
pattern, the quantity of released ssDNAs could represent that
of exosomes. Particularly, ssDNA was composed of a
combination and recognition part which is complementary to
aptamer and HP1, respectively. HP2 is a molecular beacon
designed based on the Förster resonance energy transfer
(FRET) effect between the quencher (BHQ) and fluorescent
dye (FITC) at separate ends of the hairpin structure (Figure
1c). Owing to the stronger affinity, HP2 took place of ssDNAs
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to form the HP1−HP2 complex thereby turning on the
fluorescence signal. Exact target ssDNAs allowed the initiation
of the amplification process and produced detection signals.
Exosomes separated by magnet were further purified by
Endonuclease EcoRI corresponding to restriction sites
designed in aptamers, avoiding the drawbacks of irreversible
binding of immunoaffinity capture (Figure 1d).
Characterization of Superparamagnetic Conjunc-
tions. To validate the formulation of SMCs, FTIR absorbance
was detected to prove the formation of amido bonds between
nanoparticles and aptamers. At the same time, the absorbance
intensity at 260 nm was used to quantify the aptamers before
and after amide reaction in the supernatant, an indirect
quantification method of aptamers combination. Polyacryla-
mide gel electrophoresis (PAGE) and absorbance detection
were utilized to identify ssDNAs hybridization efficiency. As
displayed in Figure 2a, the peak shift from 1690 to 1630 cm−1
was attributed to the formation vibration of the carbonyl (C
O) of the amido bond. And the peaks at 1380 and 3100 cm−1
were ascribed to the bending vibrations of C−N bonds and
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Figure 3. Validation of amplification platform for ssDNA detection. (a) PAGE gel of different samples in the amplification system: Lane 1, standard
DNA markers; lane 2, 1 μM of HP1; lane 3, 1 μM of HP2; lane 4, 1 μM HP1 incubated with 1 μM HP2 for 1 h. lane 5, 1 μM HP1 incubated with 1
μM of random DNA and 1 μM HP2 for 1 h. Lane 6, 1 μM of HP1 incubated with 1 μM of target DNA and 1 μM of HP2 for 1 h. (b) Fluorescence
intensity of molecular beacon in different samples corresponding to (a). (c) Relationship between target DNA concentration and fluorescence
intensity of molecular beacon. (d) Linear relationship between logarithmic target DNA concentrations and fluorescence intensity.

stretching vibrations of N−H bonds in the conjunction. All the
specific groups in the FTIR spectrum indicated the covalent
conjunction of aptamers and Fe3O4 cores. The intensity of
aptamers in the supernatant decreased after 12 h reaction,
indicating the binding of aptamers to superparamagnetic Fe3O4
cores, and the binding efficiency was about 80%, calculated
from absorbance peak value (Figure 2b). On the other hand,
Fe3O4 cores directly incubated with aptamers without
condensing agent caused no decrease in supernatant, which
suggested ignorable nonspecific attachment in the reaction
system (Figure S1). Figure 2c and d describes the hybrid-
ization of ssDNA1 and ssDNA2 with PAGE as well as a UV−
vis spectrograph. The bands of lane 2 and lane 3 refer to two
ssDNA standards before reaction, while lane 4 and lane 5 are
the supernatant after two ssDNAs react with aptamers. The
weakened signal suggested successful conjugation of ssDNAs
and aptamers. The hybridization efficiency calculated by gray
value and absorbance peak are shown in Figure S2, which
achieved similar efficiency results as approximately 70%. The
zeta potential of the superparamagnetic Fe3O4 cores was about
−9.8 mV due to carboxyl groups functionalized on the surface
of Fe3O4 particles. After modification with aptamers and
ssDNAs, the zeta potential decreased gradually to −16 and
−23 mV (Figure 2e), which may result from the negative
charge of ssDNAs. On the other hand, superparamagnetic
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particle-aptamers and ssDNAs conjunctions exhibited average
diameters around 20 nm, as evidenced by dynamic light
scattering (DLS) and TEM in Figure 2f and g,h, which indicate
that SMCs had ultrasmall diameters and the binding of
aptamers and ssDNAs led to a slight increase to Fe3O4 cores,
which are 10 nm in diameter. Superparamagnetic particles with
small size were more easily redispersed and exhibited better
solubility in aqueous solution, as well as stronger magnetism.
Instead, some commercial magnetic beads can easily
precipitate or form large complexes, which display disadvan-
tages on purification and separation operations.
Feasibility of Amplification Method for ssDNA
Detection. The quantity of exosomes in real samples might
result in extremely low concentration of replaced ssDNAs.
Thus, the DNA amplification method played a vital role in
exosome detection. Taking the advantages of base pairing
principle and stem-loop structure of DNA realized the
nonenzymatic amplification of target ssDNAs. First of all,
hairpin DNA HP1 and HP2, as well as HP1-HP2 complex
were determined by PAGE assay, which confirmed the
feasibility of the amplification strategy. In the absence of
target ssDNAs, no HP1-HP2 complexes were formed, and only
target ssDNAs could trigger the amplification circle. Random
DNAs were set as contrast group (Figure 3a). Oligo Analyzer
3.1 was used to calculate the relevant parameters T m
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Figure 4. Detection of PCa related exosomes. (a) Dilution and calibration curve of exosomes from LNCaP cells diluted by exosome-free urine and
PBS. (b) Linear relationship between fluorescence intensity and logarithmic exosomes count. (c) Fluorescence intensity of exosomes from different
cell cultures medium. (d) Western blot of PSMA expression in exosomes from different cell cultures medium. (e) TEM image of SMC-MB
captured exosomes.
(annealing temperature), ΔG (Gibbs free energy), ΔH
(enthalpy change), and ΔS (entropy change) of hairpin probes
HP1 and HP2 (Table S2), and predicted the stability of
oligonucleotides. As displayed in Figure S3, the hairpin
structures HP1 and HP2 were stable enough at 37 °C with
hybridization of 14 and 11 pairs of bases in the stem parts. The
Tm values of HP1, HP2, and HP1−HP2 duplex calculated by
RNA structure were 73.3, 69.0, and 71.6 °C (CNa+ = 25 mM,
CMg2+ = 4.5 mM), respectively, indicating the stability of HP1
and HP2. Furthermore, the hybridization between bases in
single DNA was superior to that of bases from different DNAs.
Additionally, the mixture of HP1 and HP2 could regain the
fluorescence signal as temperature reached 50 °C, revealing the
temperature should be carefully controlled (Figure S4). The
optimized incubation time and ratio are further discussed in
Figures S5 and S6, which was 1 h for incubation with a ratio of
1.5:1 for HP1 and HP2. To simplify the detection method of
amplified ssDNAs, HP2 was designed as a molecular beacon,
which had no fluorescence signal in the stem-loop structure
owing to the FRET between the fluorescent group FITC and
the quenching group BHQ, while it regained fluorescence in
the HP1−HP2 complex because of the remoteness of the two
groups. Similarly, the relevant fluorescence at the wavelength
of 525 nm indicated the quantity of the HP1−HP2 complex
and was presented by target DNAs (equimolar mixture of
ssDNA1 and ssDNA2). The fluorescence responses of different
samples are described in Figure 3b. Obviously, there was a
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linear relationship between logarithmic target ssDNA concen-
tration and fluorescence intensity (Figure 3c, d), demonstrat-
ing the effectiveness and practicability of the amplification
method in ssDNA detection.
PCa Related Exosomes Detection Efficiency by SMC-
MB. Based on the optimized conditions, we developed and
characterized an analysis platform for PCa related exosomes
with the PSMA aptamer. To investigate detection efficiency of
the novel platform, LNCaP cell derived exosomes were
enriched by ultracentrifugation and gradient diluted to exact
concentrations with exosome-free urine and PBS separately. As
shown in Figure 4a, the fluorescent signal elevated along with
the increasing exosome concentrations, which resulted from
increasing ssDNAs released from the SMCs. Similar to the
relationship between the concentration of ssDNAs and the
fluorescence signal of HP1−HP2 complexes (Figure 4b), a
linear relationship was obtained between fluorescent signal and
the logarithmic exosomes counts ranging from 103 to 108 /μL,
with a correlation coefficient (R2) of 0.989. Owing to the
interference of urine itself, the limit of detection (LOD) was
estimated and calculated to be ∼100 particles/μL, much higher
than the detection limit in PBS solution (∼50 particles/μL)
due to the complex urine background. However, in accordance
with the results of several studies and our research, the
concentration of urine exosomes is far beyond the calculated
LOD in this experiment, revealing the applicability in urine
exosomes detection. Furthermore, it should be noted that the
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Figure 5. Comparison between ultracentrifugation and SMC-MB platform. (a) TEM image of exosomes purified by SMC and restriction enzyme.
(b) TEM image of exosomes isolated by UC. (c) Particle diameter of SMC-MB and UC separated exosomes determined by DLS. (d) Total protein
amount and particle concentration of exosomes separated by SMC-MB and UC. (e) Specific protein expression of exosomes separated by SMC and
UC.

Figure 6. Analysis of urine exosomes in PCa patients and healthy donors. (a) TEM images of immunogold labeled exosomes from PCa patients
and (b) healthy donors. (c) Specific protein expression of exosomes separated by SMC-MB in urine of PCa patients and healthy donors. (d)
Histogram and (e) box plots for fluorescence intensity of exosomes from PCa patients and healthy donors detected by SMC-MB platform. (f) PSA
and (g) PCA3 mRNA expression quantified by qRT-PCR in PSMA-positive exosomes. (*p < 0.05; **p < 0.01; ***p < 0.001).

dual function platform described here is simple and highly
efficient without complicated instruments and expensive
reagents. To further explore the specificity of the platform,
five cancer cell derived exosomes were incubated with SMC
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and detected by MB amplification cycle. The fluorescence
intensity of exosomes from human prostate cancer cell LNCaP
was significantly higher than that of other cancer cells A549,
T24, HCT116, and HepG2 (Figure 4c), consistent with the
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protein expression levels of PSMA (Figure 4d), which also
proved the aptamer applicability in this platform. Figure 4e
displays typical binding morphology of SMCs and exosomes,
indicating successful capture of exosomes by SMCs.
Exosome Isolation Efficiency Compared to Ultra-
centrifugation. With the benefit of the restriction site of
aptamers, exosomes captured by SMCs could be easily
separated from particles. And the morphology and size
distribution of exosomes isolated by SMCs followed by
enzyme restriction were determined by TEM and DLS.
Considering that ultracentrifugation (UC) is the gold standard
isolation method in current exosome studies, which is
employed by over 50% of researchers, an ultracentrifugation
group was utilized to compare with SMC-MB platform to
further illustrate capture capacity. Thus, LNCaP cell derived
exosomes were isolated by UC, and further resuspended in
exosomes-free urine followed by SMC-MB treatment. Gen-
erally, UC and SMCs isolated exosomes were both cap-shaped
with average diameters of 30−150 nm (Figure 5a, b).
However, exosomes in UC precipitate inevitably existed with
protein aggregations and extracellular vesicles (EVs, more than
200 nm in diameter), leading to a complex background and
larger diameter determined by DLS (Figure 5c). On the
contrary, exosomes obtained by the SMCs isolation method
were observed to have fewer impurities and smaller size. Also,
the total protein and particle concentration of exosomes were
analyzed by BCA protein determination and NTA in both UC
and SMC-MB groups. As a result, more protein was acquired
by UC (Figure 5d). It may be induced by protein bundles and
EVs resulting from a nonspecific isolation method. Meanwhile,
there was no big difference of particle concentration between
two groups. Exosome-specific proteins PSMA, CD63, and CD9
detected by Western blot further confirmed the successful
exosomes isolation (Figure 5e) ,displaying the exosomes
isolation capability of the SMC-MB platform.
PCa Detection Efficiency via SMC-MB Platform.
Considering the platform exhibits excellent properties of
exosome isolation and detection, SMC-MB was further applied
to capture and detect exosomes related to prostate cancer
(PCa) in urine samples. Prostate specific membrane antigen
(PSMA) was regarded as a specific PCa biomarker in a number
of research studies, with higher expression in prostate cancer.
First, immunogold labeling of PSMA in exosomes from urine
samples of healthy donors and prostate cancer subjects is
demonstrated by TEM in Figure 6a and b, respectively.
Apparently, PSMA antibody labeled gold nanoparticles
anchored on some of the exosomes from PCa patients while
being irregularly distributed in cancer-free samples. Accord-
ingly, the SMC-MB platform mentioned above was directly
applied to analyze PSMA-positive exosomes in urine samples
of PCa patients and healthy donors, and the diagnosis
efficiency of PCa was evaluated. Exosomes separated by the
SMC platform also matched the established exosomal
morphology and size. The PSMA, CD63, and CD9 protein
expressions were determined to explain the validity of the
SMC-MB platform. It was obvious that PSMA-positive
exosomes also harbored exosomes specific proteins. However,
since our platform was designed for PSMA positive exosomes,
which was extremely low in healthy donors, specific protein
expressions of exosomes captured by SMC in healthy urine
were hard to detect as shown in Figure 6c. To further explore
the efficiency of PSMA-positive exosomes in urine for PCa
diagnosis, we collected urine samples from 20 PCa patients
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and 50 healthy donors. The fluorescent signal of the PCa
group obtained from the SMC-MB detection platform was
significantly higher than that of the healthy group (P < 0.05),
indicating the quantity of PSMA-positive exosomes in urine of
PCa patients discriminated from that of cancer-free donors
(Figure 6d, e). In addition, the total exosomes concentrations
showed no significant difference between healthy donors and
PCa patients detected by NTA (particle diameter: 30−150 nm,
Figure S7). However, the PSMA-positive exosomes concen-
tration isolated by SMC-MB was much higher in the PCa
group than in the healthy group. Therefore, PSMA-positive
exosomes reveal their potential as PCa biomarkers. In
consideration of the accepted biomarkers in clinical prediction
of prostate cancer including PSA and PCA3, qRT-PCR was
applied to detect PSA and PCA3 mRNA levels in exosomes
isolated by our platform. As a result, PSA mRNA signal
presented in 18 PCa cases and PCA3 mRNA signals presented
in 19 PCa cases (Figure 6f, g), indicating the exosomes
captured by SMC-MB were prostate cancer related. These
findings proved that higher specificity was achieved in the
newly developed platform, and the molecular signatures of
individual exosomes were also beneficial in PCa diagnosis,
which further confirmed the prospective diagnosis value of
PSMA-positive exosomes for prostate cancer.
■
CONCLUSION
A dual functional platform for exosomes detection and
isolation is presented. In this paper, we introduce an
aptamer-based strategy to separate exosomes from urine
samples and convert exosomes detection into ssDNA detection
by virtue of a signal amplification method. In the amplification
cycle, two hairpin DNAs were successfully designed to produce
a stronger signal. Consequently, the superparamagnetic
conjunction-molecular beacon (SMC-MB) platform embodied
profound capture capacity and detection efficiency, thereby
achieving ideal effects in urine exosome analysis. The LOD of
SMC-MB in urine samples was as low as 100 particles/μL,
which was competitive in current urine exosomes detection
methods.33 Compared to ultracentrifugation, SMC-MB iso-
lated exosomes were purer, containing fewer proteins and EVs.
Most importantly, the PSMA aptamer based SMC-MB
platform was useful to separate PSMA positive exosomes
from urine, which is highly expressed in prostate cancer
patients, and possesses great value in prostate cancer detection.
In summary, the SMC-MB platform is a promising method for
exosome studies and cancer diagnosis.
■ EXPERIMENTAL SECTION
Preparation of Superparamagnetic Conjunctions (SMCs).
Carboxyl-group-functionalized superparamagnetic Fe3O4 nanopar-
ticles (50 μL, 4 mg/mL) were incubated with 3-ethylcarbodiimide
hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium (NHS)
at a molar ratio of 1:2:3 at room temperature for 1 h. Next, the
activated superparamagnetic Fe3O4 nanoparticles were purified via
magnetic separation and resuspended in 200 μL of diethyl
pyrocarbonate (DEPC) treated water. Then, 10 μL of 0.5 mM
amino-modified aptamer was added in the solution and incubated for
12 h with gentle shaking. After that, the mixture was purified by
magnetic separation and washed three times. Afterward, 10 μL of 0.5
mM complementary ssDNA1 and ssDNA2 was added successively
after magnetic purification and incubated at room temperature for 2 h.
Finally, the resuspended solution (200 μL) was stored at 4 °C after
magnetic separation.
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Cell Culture and Exosomes Extraction. Exosome-free fetal calf
serum (FBS) was prepared by ultracentrifugation at 120 000g at 4 °C
overnight. LNCaP cells were cultured in RPMI-1640 medium
(Invitrogen) and A549, T24, HCT116, and HepG2 cells were
grown in DMEM medium. Both media were supplemented with 10%
exosome-free FBS, (Gibco), penicillin (100 μg/mL), and streptomy-
cin (100 μg/mL). All cells were maintained in an incubator with 5%
CO2 at 37 °C. The supernatant was first centrifuged at 300g for 10
min, 2000g for 20 min, and 10 000g for 30 min sequentially, in order
to remove cells, cellular debris, and large extracellular vesicles.
Enriched exosome samples were diluted to different concentrations
with PBS and exosome-free urine.
Urine Samples Collection. Exosome-free urine was prepared by
ultracentrifugation at 120 000g at 4 °C overnight. According to the
experimental implementation plan, and the norms and reasonable
operating procedures of collection, the whole process strictly followed
the relevant ethical requirements and all volunteers signed the
informed consent before sample collection. Twenty patients with
locally advanced PCa provided 10 mL urine in mid-morning or late
morning. Fifteen healthy male volunteers samples were collected from
the China Pharmaceutical University students and teaching staff
whose physical examination and biochemical test results showed no
exception.
Isolation and Detection of Exosomes. 100 μL of SMCs was
added into 10 mL of exosome samples at room temperature for 2 h
with gentle shaking to release ssDNAs. The SMCs captured exosomes
on their surfaces were separated by magnets and redispersed in 100
μL of PBS. Then 10 μL of HP1 (100 μM), 15 μL of HP2 (100 μM),
equivalent 0.5× Nt.BstNBI buffer, and 1× ThermoPol buffer were
introduced to 50 μL of supernatant and incubated at 37 °C for 1 h at
37 °C for 60 min. The fluorescence of molecular beacon was
measured by using a fluorescence spectrophotometer. The SMC
suspension with exosomes was mixed with 0.03 U/μL EcoRI
endonuclease in Nt.BstNBI buffer and incubated at 37 °C for 30
min. Finally, extra superparamagnetic Fe3O4 nanoparticles were
removed by magnets while purified exosomes were left in the
supernatant and kept at −80 °C for further study. Ultracentrifugation
(UC) treated samples were centrifuged at 120 000g for 2 h and
resuspended with 100 μL of PBS.
Western Blot. Purified exosomes were mixed with loading buffer
and heated to 100 °C for 15 min with loading buffer, and Western
blot analysis was carried out on 8% SDS−PAGE followed by
electrotransfer onto nitrocellulose filters. After blocking for 1 h in TBS
with 0.1% Tween 20 (TBST) and 5% skim milk, the membranes were
incubated overnight with specific primary antibody in TBST
containing 5% BSA. Detection was carried out by the use of a
HRP-labeled secondary antibody (Bioworld Technology) and
developing agent (Thermo). Western blot was scanned by Quantity
One Imaging Software from Bio-Rad.
Measurement of Exosome Size and Concentration. The
particle size and concentration were determined by dynamic light
scattering (Litesizer 500, Anton Paar) and with a NanoSight
instrument (TM LM10-HS, Malvern). The exosomes samples were
diluted with PBS to comply with the best working range of the
analysis software. Next, the diluted samples were injected into the
laser pool. The mode size, mean size, and particulate concentration in
each 1 mL sample were analyzed by NTA 3.0 analysis software. All
the measurements were performed for 60 s and repeated in triplicate.
Total RNA Isolation, cDNA Synthesis, and qRT-PCR. Total
RNA was extracted from the exosomes using the Trizol Plus RNA
purification kit (Life Technologies) according to manufacturer’s
protocol. The detection included reverse a transcription (RT) process
and quantitative real time polymerase chain reaction (qRT-PCR)
process. The RT process was conducted by the following method:
4 μL of 5× RT buffer, 1 μL of dNTP Mix (10 nM each), 1 μL of RNA
inhibitor (40 U/μL), 1 μL of RT primer (20 nM), 1 μL of M-MLV
(H−) reverse transcriptase (100 U/μL), total RNA extracted from
real samples, and DEPC-treated water were mixed to a final volume of
20 μL. The RT process was conducted under the following
conditions: 45 min at 42 °C and 15 min at 70 °C. The RT products
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were stored at −20 °C. The qRT-PCR process was conducted by the
following method: 5 μL of 2× AceTaq Master Mix, 0.2 μL of universal
forward primer (10 μM), 0.2 μL of reverse primer (10 μM), 1 μL of
RT product, 0.5 μL of U-MB (10 μM), and DEPC-treated water were
mixed to a final volume of 10 μL. The qRT-PCR process was
conducted under the following conditions: 95 °C for 5 min, 35 cycles
of 95 °C for 10 s, 45 °C for 30 s, and 60 °C for 30 s. The fluorescence
signal was detected at 45 °C.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acssen-
sors.9b00621.
Sequence information on hairpin DNA probes and other
oligonucleotides, structure and relevant parameters of
hairpin DNA probes, fluorescence intensity of amplifi-
cation products (HP1−HP2 complex) with different
incubation time and ratio, quantification methods for
incubation efficiency and fluorescence intensity of
different substrates at different temperature, comparison
of total exosome concentration from healthy donors and
PCa patients (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: guengineering@cpu.edu.cn.
ORCID
Yueqing Gu: 0000-0003-1315-4618
Author Contributions
P.L. and X.Y. contributed equally. All authors contributed to
the conception of the experiments and discussion of the results
and contributed to writing the manuscript. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors sincerely acknowledge the financial support from
Natural Science Foundation of China (NSFC 81729002,
91859204, 81727804), the 973 Key Project (2015CB755504),
State key laboratory of Natural Medicines
(SKLNMZZCX201819) and the Priority Academic Program
Development of Jiangsu Higher Education.
■
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