Article

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Colorimetric Nanoplasmonic Assay To Determine Purity and Titrate

Extracellular Vesicles
Daniele Maiolo,*,∥,† Lucia Paolini,‡ Giuseppe Di Noto,‡ Andrea Zendrini,‡ Debora Berti,§ Paolo Bergese,†
and Doris Ricotta‡
†
Chemistry for Technologies Laboratory and INSTM, Department of Mechanical and Industrial Engineering, University of Brescia,
via Branze 38, 25123 Brescia, Brescia, Italy
‡
Department of Molecular and Translational Medicine, Faculty of Medicine, University of Brescia, 25123 Brescia, Brescia, Italy
§
Department of Chemistry “Ugo Schiff” and CSGI, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Florence
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*
S Supporting Information

ABSTRACT: Extracellular Vesicles (EVs) − cell secreted

vesicles that carry rich molecular information of the parental
cell and constitute an important mode of intercellular
communication − are becoming a primary topic in transla-
tional medicine. EVs (that comprise exosomes and micro-
vesicles/microparticles) have a size ranging from 40 nm to 1
μm and share several physicochemical proprieties, including
size, density, surface charge, and light interaction, with other
nano-objects present in body fluids, such as single and
aggregated proteins. This makes separation, titration, and
characterization of EVs challenging and time-consuming. Here
we present a cost-effective and fast colorimetric assay for
probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between
colloidal gold nanoplasmonics, nanoparticle−protein corona, and nanoparticle−membrane interaction. The assay hits a limit of
detection of protein contaminants of 5 ng/μL and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which
matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the
nanoparticle−protein corona in analytical chemistry.

E xtracellular Vesicles (EVs) have been identified as

mediators of intercellular communication in many
pathophysiological processes throughout the body; for this
role they have been the subject of a growing scientific interest
over the past few years.1
These cellular secreted vesicles comprise microvesicles and
ectosomes, budded from the plasma membrane with a size from
40 nm to 1 μm, and exosomes of intracellular origin with a size
from 30 to 120 nm.2,3 EVs contain cell specific cargos such as
lipids, proteins, DNA, mRNA, and miRNA4 and populate
different biological fluids (plasma, cerebrospinal fluid, urine,
saliva, etc.), making them ideal candidates for biomarker
studies.5−7
The role that EVs are believed to play in various pathological
conditions is linked to the delivery of cargos to surrounding
cells actuating a functional manipulation of the microenviron-
ment, for example tumor derived EVs can impair antitumor
immune response reducing lymphocyte activity.8 In addition
several studies have focused on the role of EVs in cancer
biology because their release is significantly altered in cancer
cells;9 in the same way their activity in intercellular
communication2 seems to be involved in neurodegenerative
disorders.2,10−12 Therefore, EVs are envisioned as captivating
means for “remote” biopsies13,14 and theranostics.15−18
Unfortunately, understanding the molecular mechanisms of
EV biogenesis, the precise identification of EV physiological
relevance and role, and the implementation of EV based
biotechnology are to date hampered by the poor physicochem-
ical knowledge of these nanosized objects, due to inherent
shortcomings of the current bioanalytical methods used for
their separation and characterization.2,3,19,20
EVs have a low refractive index and are heterogeneous both
in size and composition. In addition, the fact that protein
complexes, especially insoluble immunocomplexes, share some
critical biophysical properties with EVs − including size, surface
charge, and interaction with light − compromises their
detection and isolation. This, in turn, affects EV quantification
by conventional bioanalytical methods, such as flow cytometry,
and purification by differential centrifugation, especially in
diseases where formation of immunocomplexes is common,
Received: September 30, 2014
Accepted: February 12, 2015
Published: February 12, 2015

© 2015 American Chemical Society 4168 DOI: 10.1021/ac504861d

Anal. Chem. 2015, 87, 4168−4176
Analytical Chemistry
including autoimmune diseases, as well as hematologic
disorders, infections, and cancer.19,21
Recently, the problem has been tackled from the analytical
side by dedicated resistive pulse sensing22 and multiplex
systems.13,14 In parallel, unedited biophysical methods,
including nanoparticle tracking analysis, Raman microspectro-
scopy, micronuclear magnetic resonance, and small-angle X-ray
scattering (SAXS) are under exploration.22 In the last year,
excellent results have been obtained with commercial9,23 and
nanostructured13,14 Surface Plasmon Resonance (SPR) sensors.
The present work is a nanotechnological contribution to
these efforts and reports about a cost-effective and fast
colorimetric assay for probing protein contaminants and
determining the concentration of EV preparations. The assay
working principle, sketched in Figure 1, exploits colloidal gold
Figure 1. Nanoplasmonic assay for probing by eye protein
contaminants (single and aggregated exogenous proteins, SAP) in
EV preparations. See the main text for a full explanation.
nanoplasmonics24−29 and the fact that nanoparticle (NP)
aggregation at lipid membranes30 is modulated by the presence
of a protein corona (PC31−34) around the NPs. The assay kit
simply consists of a probe solution of dispersed gold NPs of
15−20 nm size, which looks red due to the NP characteristic
LSPR (localized SPR, red absorption spectrum). After the
addition of a pure EV preparation to the colloidal dispersion,
the NPs adsorb and cluster at the EV membrane causing a red-
shift and a broadening of the LSPR (blue absorption spectrum),
thus a change of the solution color into blue. Conversely, in the
case of an EV preparation containing single and aggregated
protein contaminants (hereafter referred as SAP) the NPs
preferentially interact with those proteins to form a PC (EV
+SAP, Extracellular Vesicles + Single and Aggregated
Proteins).35 The PC both inhibits the interaction between
NPs and EVs and prevents NPs from aggregating at the EV
membrane, thus keeping unchanged the NP LSPR and in turn
the solution color to the initial red.
In this contribution we will present and discuss the proof of
concept of the assay, as well as its implementation for the
titration of pure EV preparations and the mechanism of signal
generation.
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■
Article
EXPERIMENTAL SECTION
EV Purification and Characterization. EVs were
separated from human serum by performing differential
centrifugation (DC) or DC and sucrose gradient fractionation
(SGF) in sequence, adapting recently published protocols20,21
(see scheme and methods in the SI). As demonstrated in this
subsection, the preparations obtained by DC contain single and
aggregated protein contaminants not associated with the EVs
(hereafter referred as SAP) which can be subsequently removed
by SGF. Therefore, the samples obtained through DC can be
considered as the model for SAP contaminated EV preparations
(hereafter referred as EVs+SAP), while the samples purified by
DC and SGF applied in sequence can be considered as the
model for pure EV preparations (hereafter referred as EVs).
EVs and EVs+SAP were first verified by routine biochemical
and biophysical analysis21 (materials and methods are given as
SI). Both preparations resulted in containing high concen-
trations of the same EV population, as evidenced by the
characteristic phosphatidyl-choline and sfingomyelin lipid
content, by the acetylcholine-esterase enzymatic activity by
the presence of the typical exosomal and microvesicular
markers (SI, Figure S2). The morphology of the EVs was
checked by Scanning Electron Microscopy (SEM) (SI, Figure
S2) and Atomic Force Microscopy (AFM), which gave
consistent results (SI, Figures S3 and S4). In particular, both
EVs and EVs+SAP resulted in containing vesicles of the
expected36 spherical shape and size distribution, centered at
160 nm. The nature of the considered EV populations was
further confirmed by the ζ-potential value, equal to −17.7 ± 2.3
mV for EVs, while it is lower for EV+SAP (−10.3 + 1.2 mV)
due to a higher presence of proteins in the sample that lower
the “apparent” ζ-potential.37,38 Finally the hydrodynamic sizes
obtained from Photon Correlation Spectroscopy (PCS) (Figure
2A), and the positivity to the TritonX-100 test (SI, Figure S3,
panel C), are all in perfect agreement with the typical
characteristics of purified EVs reported in the literature.19
The total protein content of EVs and of EVs+SAP derived
from 1 mL of human serum was first determined through a
Bradford quantification39 and resulted in 0.3 μg/μL for EVs and
9 μg/μL for EVs+SAP, respectively (SI, Figure S2). These
values highlight a significant difference in protein content
between the two samples. However, the Bradford assay
routinely used to quantify the total proteins and often used
to quantify EVs is biased by the lipid amount in the sample and
therefore does not allow discriminating between EV associated
proteins, SAP, and lipids.20 This drawback, recurrent in
literature,20,40 has been circumvented performing dedicated
experiments of PCS, AFM, and developing an ad hoc native
agarose gel electrophoresis assay.
PCS results are reported in Figure 2A, where the scattered
field autocorrelation curves for EVs (black triangles) and EVs
+SAP (red circles) are reported. Here EVs+SAP displays a
faster and steeper decay of the autocorrelation function, G(τ),
with respect to EVs, indicating that the EVs+SAP sample
contains a significant fraction of objects with a smaller size with
respect to the EV sample (see also SI Figure S4).
To confirm that these smaller particles are SAP, we
developed and ran an agarose gel electrophoretic assay in
native conditions for the separation of a mixed colloidal
solution.41,42 We fluorescently stained EVs derived from 1 mL
of human serum, EVs+SAP derived from 1 mL of human
serum, 3 ng of synthetic phosphatidylcholine (PCh) liposomes
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Figure 2. (A) Normalized autocorrelation curves obtained through photon correlation spectroscopy (PCS) for EV and EVs+SAP preparations. The
lower decay time of the EVs+SAP is due to the presence of SAP. (B) Agarose electrophoretic separation of the EVs and of the EVs+SAP
preparations. Left panel and right panel refer to PHK67 fluorescent staining and Comassie staining, respectively. PCh liposomes (PCh ves) and BSA
were loaded as control. (C and D) AFM images of EVs and EVs+SAP adsorbed on mica (topographic mode).

of 150 nm average diameter, and 10 μg of Bovine Serum
Albumin (BSA) protein with the marker PKH67, specific for
lipid bilayers (all diluted in 10 mM PBS solution, see the SI for
description of the experiment).
Note that PCh liposomes are a convenient synthetic mimic
of EVs, since they have similar lipid composition and size
distribution with respect to EVs;23 the PCh liposomes were
prepared as described in ref 30.
The preparations were run on the gel shown in the left panel
of Figure 2B, where dark bands corresponding to the lipid
vesicles appear at the same height in the lanes of the 150 nm
liposomes (PCh ves), EVs, and of the EVs+SAP, while they are
not present in the BSA lane. The smearing of the EVs+SAP
band can be attributed to the SAP contaminants present in the
sample. The Coomassie staining, which marks both lipids and
proteins, of the same gel in the right panel of Figure 2B
confirms this interpretation and evidence that the SAP content
is negligible in the EV sample. Therefore, the small amount of
proteins measured in EVs by the Bradford assay consists of
proteins and lipids associated with EVs.
These salient characteristics are definitively confirmed by the
AFM images of EVs and EVs+SAP deposited onto freshly
cleaved mica. In fact, while the EVs sample shows isolated and
well-defined EVs of 100−200 nm size lying onto the
subnanometer smooth mica surface (Figure 2C), the EVs
+SAP sample (Figure 2D) consists of a few EVs of the same
size buried into a matrix of smaller SAP nanoparticles. All these
observation confirm, as already shown,20 that EVs retrieved by
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DC or by DC-SGF share the same integrity and structure and
that the preparations just differ for the presence or not of SAP
contaminants.
Synthesis and Characterization of Au NPs. Fifteen nm
Au NPs were synthesized in Milli-Q water (nanomolar
concentration) following the classical Turkevich protocol and
characterized by UV/vis/NIR spectroscopy, AFM, and PCS.
Full details are given in the SI.
UV/vis/NIR Spectroscopy. UV−vis spectra were measured
with a JASCO UV/vis/NIR spectrophotometer. Briefly, NP or
PC were resuspended in the appropriated solvent (water or
PBS) and the absorbance of the solution measured in the
presence and absence of the different EVs preparations.
Nanoplasmonic Assay for Detecting SAP in EV
Preparations. EVs and EVs+SAP were prepared and
characterized as described in the SI and then resuspended at
the desired concentration using PBS. EVs and EVS+SAP were
then incubated for 20 min, with gold NPs (concentration 6
nM), and the spectroscopic features of the solution were then
evaluated using a JASCO spectrophotometer. Alternatively gold
NPs after their purification from a 10% human serum solution
were incubated with EVs. The same solutions were also
measured using Photon Correlation Spectroscopy using a
Brookhaven Instrument 90 Plus (Brookhaven, Holtsville, NY).
One mL of human serum was processed by DC (see Results
and Discussion). The supernatant of the solution containing
the soluble SAP was then collected discarding the pelleted
fraction containing the EVs. SAP were quantified by Bradford
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Figure 3. (A) 15 μL of the nanoplasmonic assay, that is an aqueous dispersion of 15 nm gold NPs at 6 nM concentration, upon the addition of 10 μL
of a 10 mM PBS solution containing EVs+SAP (left tube) or EVs (right tube) derived from 1 mL of human serum. (B) UV/vis/NIR absorption
spectra of the assay before and after the addition of EVs and pure 10 mM PBS. (C) UV/vis/NIR absorption spectra of the assay before and after the
addition of EVs+SAP. Spectra were acquired with 1 nm step size and a data point every 25 nm highlighted with a symbol to ease comparison. (D)
and (E) SAP progressive inhibition of NP clustering visually probed with a multiwelled plate and quantified by the NP aggregation index (AI),
respectively. In panel (E) the errors bars indicate the standard error of three different replicates.

assay and were spiked in SGF purified EVs at concentrations

ranging from 5 ng/mL to 1 μg/mL. The PBS solutions
containing increasing concentrations of SAP and EVS were
then incubated with gold NPs (concentration 6 nM). After 20
min of incubation 100 μL of each sample was loaded on a 96
wells plate, and the absorbances of the solutions were measured
by a plate reader at the wavelengths 540 and 650 nm.
Plasmonic Titration. Increasing concentrations of the
synthetic PCh vesicle were resuspended in Tris NaCl EDTA
(the same concentration used for the EV sample), and they
were then diluted in PBS (see the main text for the
concentration tested) and incubated with gold NP (concen-
tration 6 nM). After 20 min of incubation samples were
evaluated by UV/vis/NIR spectroscopy using a JASCO UV/
vis/NIR spectrophotometer.
Other Experimental Information. A fully detailed
Materials and Methods section is given in the SI, which
includes the description of chemicals and the standard methods
and techniques used (e.g., immunoassays, synthesis of PCh
liposomes, AFM and SEM microscopy, PCS experiments, and
data analysis).
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■ RESULTS AND DISCUSSION
Probing the Presence of SAP in EV Preparation by the
Nanoplasmonic Colorimetric Assay. The nanoplasmonic
assay consists of 5 μL of a 6 nM water dispersion of 15 nm gold
NPs, stabilized by citrate anions (the physicochemical
characterization has been reported in the SI Figures S5 and
S6). Figure 3A shows the assay at work. When 10 μL of a 10
mM PBS solution containing EVs derived from 1 mL of human
serum is added, the solution color turns into blue (right tube).
Conversely, upon addition of the same amount of EVs+SAP the
solution keeps the initial red color (left tube) (a thorough
report about EV and EV+SAP preparations and character-
ization can be found in the Experimental Section and in the
Supporting Information). Therefore, the assay allows for
probing EV preparation purity by eye.
The assay optical proprieties were quantitatively investigated
by UV/vis/NIR spectroscopy.43 To ensure optimal conditions
for collecting gold NPs LSPR absorption spectra, all the
analyzed assay solutions were diluted to the same final NP
molar concentration of 2 nM. From the spectra, reported in
Figure 3B, we learn that the addition of the EVs drives a red
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Figure 4. (A) UV/vis/NIR absorption spectra of PBS solutions containing a fixed concentration of NPs and increasing concentrations of EVs,
obtained after the dilution of the EV stock preparation (EVin is the dilution ratio of the stock solution). Spectra were acquired with 1 nm step size
and a data point every 25 nm highlighted with a symbol to ease comparison. (B) AI obtained from part A plotted versus EVin and linear regression fit
(red line). (C) UV/vis/NIR absorption spectra of PBS solutions containing a fixed amount of NPs and increasing concentrations of PCh liposomes.
Spectra were acquired and represented as in panel A. (D) Calibration line obtained by plotting the NP AI obtained from part C versus the PCh
liposome solution concentration (black circles) and linear regression best fit (red line, R2 = 0.99). Errors bars indicate standard error of three
different replicates. The star point highlights the intercept of the AI value of the EV preparation (obtained from the spectra of Figure 3B) with the
regression line. The star point projection on the abscissa axis allows for the extrapolation of the unknown concentration (x = 4.5 ·109 vesicles/mL).

shift (blue triangles) of the initial LSPR peak (red squares).
This well agrees with the observed color change of the solution
from red to blue and mirrors the agglomeration and clustering
of NPs at the EV surface.30 The hypothesis that the red shift is
not merely due to NP aggregation caused by the addition of 10
mM PBS solution (through charge shielding) is ruled out by a
control sample obtained by adding pure 10 mM PBS solution
to the NP solution (and by other results presented in the next
sections of the paper). The spectrum of this control sample
(black circles) has a markedly different profile with respect to
the one of EVs+NP and is characterized by the unspecific
broadening of the LSPR peak (see the SI for replicates of the
experiment). Figure 3C shows the typical LSPR spectrum
obtained upon addition of EVs+SAP to the NP solution. In
contrast to what happens with the addition of EVs, here the
LSPR peak undergoes a slight broadening and red shift with
respect to bare NPs (black circles and red squares spectra,
respectively). These features account for the unchanged color
of the solution and are typical of gold NPs cloaked by a protein
corona.44 This suggests the NPs are passivated (with respect to
aggregation or clustering) by a preferential interaction with the
SAP contained in the sample, eventually bringing to the
formation of isolated NP-protein corona (NP-PC) complexes
(see later in the text for a thorough discussion).
We then investigated the dynamic range of the nano-
plasmonic assay. Ten mM PBS solutions containing a fixed
concentration of pure EVs were spiked with increasing amounts
of SAP varying from 5 ng/μL to 1 μg/μL molar concentrations
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(10 mM PBS solution), covering the typical SAP concentration
range commonly found in EVs prepared from biological fluids
(see the “EV Purification and Characterization” section and refs
19 and 21).
The samples were then loaded in a multiwell plate, and the
probe NP solution was added (again to a final NP
concentration of 2 nM). The most relevant results are
displayed in Figure 3D. The addition of the NP probe to the
EVs with an increasing SAP amount is mirrored by a gradual
change of the solution color from blue to red, indicative of
gradual NP aggregation inhibition. NP aggregation was
quantified through the aggregation index (AI), which can be
defined for spherical gold NPs as the ratio of the LSPR
absorption peak of pure NPs and the LSPR at some significant
red-shift wavelength.19,45 In view of Figure 3B and 3C spectra,
here we chose LSPR absorption at 540 and 650 nm (AI =
A540/A650). The obtained AI values are plotted against the
SAP concentrations in Figure 3E to yield a calibration curve for
the SAP detection range of the nanoplasmonic assay, which hits
a lower limit of 5 ng/μL of SAP, far below the typical SAP
content in EVs preparations.19,21 Additional tests of the assay
effectiveness against pure EVs, high SAP concentration (4 μg/
μL), and human serum are reported in the SI, Figure S7.
Determining EV Concentration in Pure EV Prepara-
tions. The nanoplasmonic assay can be implemented to titrate
pure EV preparations (viz. preparations containing negligible
amounts of SAP). Titration grounds on the finding that in pure
EV solutions the LSPR red shift, viz. the AI, of the NPs has a
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Figure 5. (A) Normalized UV/vis/NIR spectra obtained for an aqueous solution containing 15 nm gold NP and a 10 mM PBS solution containing
the same NP coated by a human serum PC in the presence or absence of EVs. Spectra were acquired with 1 nm step size and a data point every 35
nm highlighted with a symbol to ease comparison. (B) AFM phase imaging of 15 nm gold NP and the same NP coated by a human serum protein
corona (C) after their interaction with EVs and their deposition on a mica substrate. Inset reports a magnification of the EV boxed. NP clustering
over an extended EV zone can be detected (dark area on EVs in B), while PC adsorption at the EV surface results in a limited event. (D)
Autocorrelation curves obtained for an aqueous solution of 15 nm gold NPs (NP) at nanomolar concentration, the same NP coated by a human
serum protein corona (PC) and dispersed in 10 mM PBS, 10 mM PBS solution of EVs (EVs), and a 10 mM PBS solution containing EVs+PC or
EVs+NP. The amplitude of the autocorrelation curves is normalized to 1 for better comparison of the characteristic diffusion time, τ.

linear dependence on the NP/EV molar ratio, as shown in
Figure 4A and 4B. Since the molar concentration of any EV
preparation is unknown, we used the 150 nm synthetic
phosphatidylcholine (PCh) liposomes introduced in the
Experimental Section (Figure 2B) − which are synthetic
vesicles that mimic EVs in both size and lipid composition − to
build a calibration line. PCh liposome molar concentration can
be easily calculated from the stoichiometry of the prepared PCh
solution.30
In particular, 10 mM PBS solutions of PCh liposomes with
concentrations ranging from 2.1 × 107 to 2.1 × 1010 vesicles/
mL, viz. from 35 fM to 35 pM, were prepared. This range was
chosen as it includes the pathophysiological EV concentrations
reported in the literature.46 Each PCh liposome solution was
then incubated with the 6 nM solution of 15 nm gold NPs in a
1:1 volume ratio. The collected LSPR spectra are shown in
Figure 4C. Here we see that at the highest liposome
concentration the spectrum (blue squares) is slightly shifted
with respect to one of the isolated gold NPs reported in Figure
3B and 3C, with the LSPR peak centered at about 540 nm.
Then, as the liposome concentration decreases, the LSPR peak
undergoes a gradual red shift and a decrease of intensity (red
circles). This suggests that NP clustering is linearly dependent
to the liposome concentration and becomes more and more
prominent along with the decrease of the vesicle concen-
tration.47 Interestingly the LSPR spectra of the samples
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containing the lower EV or PCh liposome concentration
(yellow triangles up and red circles, presented respectively in
Figure 4 A and C) are characterized by the presence of a
shoulder in the near IR region. This spectroscopic feature is
very likely ascribable to the presence of nanoparticles
structuring in dimers, that can couple the LSPR transverse
mode proper of isolated nanoparticles to a longitudinal mode
due to the presence of interconnected particles.48,49
By applying the calculations presented in our recent study on
the interaction of gold NPs with giant unilamellar vesicles30
(see the SI), we were able to evaluate the dependence of the
clustering to the NP cross section/EV surface area, finding that
at the highest liposome concentration the overall surface of the
NPs matches the overall surface of the external bilayer leaflet of
the liposomes and that as the liposome amount decreases the
overall surface of the NPs exceeds the liposome one up to a
factor of 10. This suggests that at the highest liposome
concentration the amount of NPs is too low to saturate the
overall liposome surface and clustering is negligible; clustering
is then progressively triggered as the liposome/NP ratio
decreases and gets to plateau for low vesicle concentration.30
Indeed, as suggested by comparison of Figure 4A and 4C,
analogous scenario and reasoning hold if one has EVs in place
of PCh liposomes.
In Figure 4D the AI from Figure 4C is plotted as a function
of the liposome concentration, and, as evidenced by the linear
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regression line, in the investigated conditions the parameters
are in linear dependence. This calibration line finally allows for
extrapolating the unknown concentration of any pure EV
solution, provided it falls in the calibration range.
For example, we determined the concentration of the EV
preparation purified from human plasma used throughout this
work. The AI value was obtained from the UV/vis/NIR
spectrum reported in Figure 3B. In particular the star point in
Figure 4D highlights the intercept of the AI value with the
regression line, namely x = 4.5 ·109 vesicles/mL. Since the
original EV sample was diluted to 1:12 vol/vol in a 10 mM PBS
solution for UV/vis/NIR spectrometry, from the value of x and
of the dilution proportion we obtain the original concentration
of the sample is equal to 5.4 × 1010 vesicles/mL.
Remarkably, this value is consistent with the typical EV
concentrations in human plasma.20
Titration was successfully replicated on another EV
preparation separated from a different donor plasma and by
using a different batch of NPs and PCh liposomes, proofing the
robustness and repeatability of the titration method (SI, Figure
S8).
Further Assessment of the Interaction between NPs,
SAP, and EVs. The (bio)chemical/(bio)physical mechanism
we propose for explaining the signal generation of the
nanoplasmonic assay (sketched in Figure 1) relies on the
extension to EVs of our recently reported findings about the
interaction of gold NPs with synthetic Giant Unilamellar
Vesicles.30 In essence, our experimental evidence are in
agreement with the claim that bare (citrate capped) gold NPs
cluster at the EV surface, while the formation of a protein
corona (PC)31 around the NPs strongly attenuates this
phenomenon − this for NP concentrations in excess with
respect to the full coverage (upon adsorption) of the EV
external bilayer leaflet, see ref 30 for a thorough discussion.
To substantiate this hypothesis, we compared the interaction
of the assay NPs and the same NPs passivated with a protein
corona from human serum (hereafter referred as PCs) with
EVs. Figure 5 shows the UV/vis/NIR spectra of a 2 nM water
solution of bare gold NP (red squares), of a 2 nM PBS solution
(PBS 10 mM) of PC (blue triangles), and of the PC solution
after incubation with a 10 mM PBS solution of EVs of
concentration of 4.2 × 109 vesicles/mL EVs (black circles).
All three spectra share the same main features, showing a
LSPR peak centered at 540−550 nm, indicating that all the
samples contain isolated NPs. Remarkably, the spectra of the
PC+EV sample (gray triangles) definitively matches the one
obtained for the NP+EV+SAP sample reported in Figure 3C.
This indicates SAP inhibits the interaction between NPs and
EVs as a protein corona formed ex situ does.
Interaction between EVs and PC was further confirmed
through an AFM morphological analysis of EVs+NP and EVs
+PC deposited on mica. As the AFM phase image of EVs+NP
shows (Figure 5B), the presence of areas of the lipid bilayers
with different composition and stiffness, visible as dark clusters
that decorate the EV membrane (see inset), is consistent with
the presence of NPs onto the membrane.
Conversely, for the PC+EV sample (AFM phase image,
Figure 5C) the presence of the PC limits the formation of NP
clusters, as most of the PC can be found on the background of
the mica surface, and very few NPs can be visualized adsorbed
at the EV surface (inset).
To further prove this hypothesis in situ, we performed some
equilibrium binding experiments with photon correlation
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Article
spectroscopy.50 Applying this method we were able to measure
the diffusion coefficients of the NP and of the EV+NP sample,
that are related to the hydrodynamic sizes of the diffusing
objects through the Stokes−Einstein relationship. The average
of 30 autocorrelation curves for the NPs before and after the
formation of the PC have been evaluated. Figure 5D shows that
the characteristic diffusion time of NPs increased in the
presence of PC. In particular, PC formation drives a shift of the
autocorrelation curve to higher decay times τ, while when PC
are dispersed in the presence of EVs there is a small increase of
the decay time that can be attributed to a partial interaction of
PC with EVs. On the contrary the clustering of NP at the EV
surface drove a higher shift of the autocorrelation curve that is
similar to the one obtained for EVs. This hints to the presence
in solution of object containing NPs with size similar to the size
of the EVs. The ξ-pot for these hybrid NP-EV objects is
different (−11.1 ± 1.3 mV) from the one of the NPs alone in
water (−31.6 ± 3.3 mV) or the NPs dispersed in PBS (−24.8 ±
0.5 mV) (see SI Figure S9) suggesting the presence of NP-
membrane complexes than simple NP aggregates. Moreover
the fact that NP clustering at EV membrane, that is mirrored by
a variation of the AI, has a linear dependence on the EV
concentration in the studied range (see Figure 4 A-D)
represents another genuine control of the ability of the assay
to sense EVs in pure EV preparations.
Taken together, all the above data point to the mechanism of
signal generation of the assay that is summarized and described
in Figure 1. In the presence of pure EV preps, NPs can freely
interact with EV membranes clustering at their surface, while in
the presence of SAP the NP preferentially interact with them
forming a SAP corona which inhibits membrane clustering.
Therefore, NP-biomolecule interactions can be exploited as
nanoplasmonic transducer of the presence of SAP or as
nanoplasmonic gauge for the evaluation of the EV concen-
tration.
■ CONCLUSIONS
We presented a nanoplasmonic colorimetric assay for probing
by eye the presence of SAP contaminants in EV preparations
and to determine the concentration of EVs in pure
preparations. The assay applies to the whole multiscale
population of EVs in body fluids, which comprises vesicles
from 40 nm to 1 μm in size.
The working principle of the assay is based on the fact that
gold NP clustering at the EV membrane and the related LSPR
shift is directly gauged by the amount of SAP contained in the
preparation. Bare gold NPs adsorb and aggregate at the EV
membrane in pure EV preparations, while this phenomenon is
inhibited in SAP contaminated preparations by the SAP-NP
interaction that leads to the formation of a passivating protein
corona that keeps the NPs dispersed, inhibiting NP-EV
interactions. The two conditions are optically characterized
by a specific LSPR shift, which therefore acts as a color switch,
giving a naked eye colorimetric read out of the presence of
protein contaminants, that is visible down to 5 ng/μL of SAP,
far below the typical concentrations of SAP in EVs
preparations.
Remarkably, the LSPR red-shift driven by NP clustering at
the EV membrane is directly proportional to the EV
concentration. This discloses the opportunity to exploit the
assay for titrating EV pure preparations, which is another
fundamental issue in EV sample analysis.20 The titration
method resulted robust and repeatable, with a dynamic range
DOI: 10.1021/ac504861d
Anal. Chem. 2015, 87, 4168−4176
Analytical Chemistry
from 35 fM to 35 pM, matching the range of EV concentration
in body fluids.
These findings are an effective application of nanotechnology
in biology with immediate impact in EV biology − which strives
for effective analytical procedures that fit the requirements of
low volumes, reduced operative costs, fast readout, and
compatibility with common laboratory practices51,52 − and
complement the available information on the interaction of
NPs with biological membranes.53 On a wider perspective, the
fact that the assay applies invariably to micro- and nanovesicles
prompts its extensions to other biomembrane objects, such as
membrane coated viruses or organelles isolated from cell
materials.
Finally, it is worth noticing that this work widens to clinical
and biological analytical chemistry applications aimed at
exploiting, rather than avoiding, the NP-protein corona, that
to date, with few exceptions,59 have been focused on drug
loading and delivery.54−58
■
ASSOCIATED CONTENT
*
S Supporting Information
Supplementary data, figures, and a fully detailed Materials and
Methods section. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: daniele.maiolo@polimi.it.
Present Address
∥
European Center for Nanomedicine CEN foundation, c/o
Nanostructured Fluorinated Material laboratory, Department of
Chemistry, Material and Chemical Engineering, Milan Italian
Polytechnic, Via Mancinelli 7, 20131 Milan, Italy.
Author Contributions
D.R. selected and collected serum for EV purification. D.M.,
L.P., A.Z., and G.D.N. performed the experiments, with the
exception of PCS experiments, that were performed by D.B.
D.M., D.R., and P.B. conceived and supervised the work. 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 would like to thank Sara Busatto for technical
assistance, Kimberly Hamad Schifferli, Francesca Baldelli
Bombelli, Ciro Chiappini, and Eugenio Monti for fruitful
discussions and suggestions. We thank Marco Vitale and
Davide Dallatana for SEM consulting and Roberta Giuliani for
use of a UV−vis spectrometer. Dr. Di Noto received a
fellowship “Bando Mecenati” from “Fondazione CEUR”. D.M.
is also grateful to the Academia Delle Nanoscienze di Gagliato
(Nanogagliato) for the Salvatore Venuta fellowship. This work
was supported by MIUR (Ministero Italiano dell’Università e
della Ricerca) through the project Soft Matter Nanostrutturata:
dall’indagine chimico-fisica allo sviluppodi applicazioni innova-
tive, PRIN 2010−2011 grant 2010BJ23MN to D.M., D.B., and
P.B. and by University of Brescia Research found (ex 60%) and
“‘Prima spes onlus”’ foundation to D.R., L.P., A.Z., and G.D.N.
4175
■
Article
ABBREVIATIONS
NP nanoparticle
PC protein corona
EVs extracellular vesicles
SAP single and aggregated protein contaminants
DC differential centrifugation
SGF sucrose gradient fractionation
SPM scanning probe microscopy
■
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4176 DOI: 10.1021/ac504861d

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