Article

Cite This: Anal. Chem. 2019, 91, 2224−2230 pubs.acs.org/ac

Ultrasensitive Electrochemical Immunoassay Based on Cargo

Release from Nanosized PbS Colloidosomes
Xiujuan Han, Hongfang Zhang,* and Jianbin Zheng*
Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry and
Materials Science, Northwest University, Xi’an 710127, China
*
S Supporting Information

ABSTRACT: Colloidosome is a novel nanostructure com-

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posed of millions of colloid particles. In this work, nanosized

PbS colloidosomes were initially prepared and applied as
nanoprobes for an ultrasensitive immunoassay. The colloido-
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somes were simply prepared in mild conditions by assembling

the elementary approximately 8 nm PbS nanoparticles at the
water-in-oil interface of emulsion droplets. To enhance the
rigidity and biocompatibility of the colloidosomes, interfacial
polymer was introduced by utilizing self-polymerization of dopamine. By treating with dilute nitric acid, a bursting release of
lead ions from the colloidosomes occurred and the lead ions can be detected easily by anodic stripping voltammetry. In this
way, a colloidosome-based electrochemical immunoassay was developed by using the nanosized PbS colloidosomes as
electroactive labels. The proposed method featured a linear calibration range from 10 fg·mL−1 to 100 ng·mL−1 with a low
detection limit of 3.4 fg·mL−1 for the detection of human epididymis protein 4. This work introduced a new member for the
family of colloidosomes and offered a novel perspective for the rational implementation of various colloidosomes for novel low-
abundance cancer biomarkers analysis.

C olloidosomes (CSs), first termed by Dinsmore et al. at

2002,1 refers to solid and hollow capsules composed of
colloid particles and usually prepared by self-assembly of
colloid particles on emulsion droplets. As witnessed by the
pioneering studies, the initial interest for CSs was their
important internal volume combined with the properties of the
nanobuilding blocks in the shell, which facilitated the
capsulation of active ingredients such as drugs, flavors,
proteins, bacteria, or even living cells.2−5 Different with the
simplex phospholipid bilayer capsules of liposomes, the
nanobuilding blocks of CSs are theoretically unlimited. As a
hollow structure,6−12 the packing colloidal particles of CSs
are a wide variety of nanoparticles (NPs) including iron oxide,6
gold,7 silver,8 polymer,9 silica,10 metal−organic frameworks,11
and the combination of these NPs.12 As a matter of course, the
specific physical properties of CSs as a novel nanostructure
were noticed. For instance, hollow core−shell α-Fe2O3 CSs
were evaluated as a lithium-ion battery anode13 and carbon
CSs were demonstrated as good electrocatalysts for oxygen
reduction reaction.14
The identification and detection of disease biomarkers have
become important issues for the early disease diagnosis.15,16
Electrochemical immunoassay is a sensitive technique for the
early disease diagnosis through the accurate determination of
specific protein biomarkers at ultralow levels. To quantitatively
transform the immuno-recognition event into sensitive electro-
chemical signals, different tracing nanoprobes were designed.17
Among them, nanoparticles of metal (e.g., Cu, Ag) or metal
sulfides (e.g., CdS, PbS) are frequently applied because of their
intrinsic redox properties. Wang’s group18 pioneered this
concept by using ZnS, CdS, PbS, and CuS colloidal crystals for
the simultaneous analysis of four proteins. These nanocrystal
labels exhibited detect limit (DL) of ∼10 ng·mL−1 for β2-
microglobulin, IgG, bovine serum albumin (BSA), and C-
reactive protein. Using the same concept, Chen et al.19
obtained DL of 3.4 pg·mL−1 for the detection of IgG1 with
CdS quantum dot-doped BSA as the signal tracing nanoprobes.
Both the studies applied the magnetic separation and
voltammetric measurement procedures. Thus, the about 10-
fold increase of the average size of the CdS nanoparticles
utilized in the latter study owed much to the dramatic decrease
of DL (10 ng·mL−1 vs 3.4 pg·mL−1) when compared with that
obtained by Wang’s group.18 Zhu’s team20 ingeniously
designed a sandwich type electrochemical immunoassay by
using the rolling circle amplification products as the specific
templates for the subsequent Cu NPs formation. The DL for
prostate specific antigen was as low as 0.02 fg·mL−1 because
cascade signal amplification was easily achieved by dissolving
the CuNPs and detecting the released copper ions.
Quite understandably, the immunoassay sensitivity is greatly
connected with the metal ions concentration that the metal-
containing nanocrystal tracers can supply. Of course, this does
not mean that superlarge tracers are needed. Large tracers
definitely create steric hindrance that causes poor antigen−
antibody binding interaction and inversely affect the perform-
Received: October 19, 2018
Accepted: January 9, 2019
Published: January 9, 2019

© 2019 American Chemical Society 2224 DOI: 10.1021/acs.analchem.8b04807

Anal. Chem. 2019, 91, 2224−2230
Analytical Chemistry
Scheme 1. Schematic Illustration of the PbS CSs-Based Electrochemical Immunoassay
ance of immunoassay. From this point of view, nanosized CSs
are excellent candidates for signal tracers of electrochemical
immunoassay because of the tunable size and rich metal
component distributed at the nanobuilding shells. According to
the calculation of Duan et al.,21 one Fe3O4 colloidosome was
composed of about 6.1 × 108 nanoparticles (8.0 nm of average
size). This means that if the metal CSs are utilized as the
tracing nanoprobes for electrochemical immunoassay, the
cargo release of the CSs shell would give rise to a “burst” of
metal ions. And the electric signal will be amplified
exceptionally when compared with it using the basic metal
NPs as tracing nanoprobes. Inspired by this exciting
consideration, we try to develop a proof-of-concept colloido-
some-based electrochemical immunoassay.
Herein, the nanosized, polydopamine-coated PbS CSs were
initially prepared by a reverse water-in-oil emulsion system.
And an ultrasensitive immunoassay based on the PbS CSs was
developed. As shown in Scheme 1, the functional fullerene-
chitosan (C60-Chit) nanocomposite was immobilized onto a
glassy carbon electrode (GCE) to covalently immobilize the
capture antibodies and expectedly improve the detection
sensitivity.22 Human epididymis protein 4 (HE4), a tumor
marker of great importance for its potential to reflect the
differentiation stage of the ovarian carcinoma,23 was chosen as
the model target. After the PbS CSs was introduced onto the
electrode surface by the formation of the sandwich-type of
immunocomplex, Pb2+ released from the CSs was detected. As
a result, the developed immunoassay exhibited a ultralow
detection limit of 3.4 fg·mL−1. The strategy offered a novel
perspective for the rational implementation of CSs for HE4
detection and could be integrated with other recognition
elements to broaden its applications in bioassay.
■ EXPERIMENTAL METHODS
Reagents and Materials. Oleylamine (OLA, 80−90%),
PbCl2 (99.999%), and glutaraldehyde (GA) were purchased
from Aladdin. HE4, monoclonal anti-HE4 antibody (Ab1) and
polyclonal anti-HE4 antibody (Ab2) were obtained from Linc-
Bio Science Co. Ltd. (Shanghai, China). BSA was obtained
from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijing,
China). 3-Mercaptopropionic acid (MPA, 99%) was purchased
2225
Article
for local reagents companies and used as received. All other
chemicals were analytical grade. Phosphate buffered saline
(PBS, pH 7.4) was prepared by mixing Na2HPO4 and
KH2PO4. PBS containing 0.05% Tween-20 (PBST) was used
as the washing solution.
Characterization. Scanning electron microscopy (SEM)
images were obtained by using a JSM-6390A scanning electron
microscopy (JEOL). The transmission electron microscopy
(TEM) images were determined using a Tecnai G2 F20 S-
TWIN (FEI, U.S.A.). All powdered X-ray diffraction (XRD)
experiments were recorded on a D8 ADVAHCL* diffrac-
tometer (Bruker) using Cu Kα radiation. Inductively coupled
plasma optical emission spectrometry (ICP-OES) was carried
out on an Optima 2100DV (PerkinElmer). All voltammetric
experiments were carried out with a CHI660E electrochemical
workstation (Chenhua Instruments). A conventional three-
electrode system with either a bare or modified GCE as the
working electrode, a platinum electrode as the counter
electrode and a saturated calomel electrode as the reference
electrode was applied.
Preparation of the PbS CSs. In typical experiment, 10 mg
of PbS NPs was dispersed in 2 mL of oil phase, containing 50
vol % vegetable oil and 50 vol % heptane under ultrasonication
for 10 min. Then, 400 μL of Tris-HCl buffer (10 mM, pH 8.5)
was added to the PbS NPs dispersion (the volume ratio of the
oil to water was 5), and the two-phase system was sonicated
for 1 h in an ultrasound bath to generate homogeneous water-
in-oil emulsions. The mixtures were left unperturbed for 1 h
after sonication. After the formation of a Pickering emulsion,
0.40 mg dopamine powder (DA/Tris-HCl = 1 mg·mL−1) was
added to the emulsion, followed by ultrasonication for 10 min
and left unperturbed for 10 min, giving rise to a dark gray
emulsion composed of a large number of PbS CSs. In order to
transfer the CSs from the organic to the aqueous phase, the
system was centrifuged for 5 min at 3000 rpm. After
centrifugation, the heptane phase was carefully removed from
the centrifugation tubes, and the aqueous phase containing the
CSs was collected.
Preparation of PbS CSs Based Nanoprobe. A total of
100 μL of the dispersion of the as-synthesized PbS CSs was
mixed with 100 μL of pH 7.4 phosphate buffer. Then, 2 μL of
DOI: 10.1021/acs.analchem.8b04807
Anal. Chem. 2019, 91, 2224−2230
Analytical Chemistry Article

Figure 1. Preparation and characterization of PbS CSs. (A) Schematic illustration of the preparation of PbS CSs. (B−D) SEM images of PbS CSs.
Scale bar is 1 μm for B, 400 nm for C, and 100 nm for D.

Figure 2. SEM images of PbS CSs prepared with different CDA(A−C), mPbS (D−F), RO/W (G−I), and oil phase (J−L). Scale bar is 500 nm for A−F
and 1 μm for G−L.

2226 DOI: 10.1021/acs.analchem.8b04807

Anal. Chem. 2019, 91, 2224−2230
Analytical Chemistry
Figure 3. Pb2+ release and detection. (A) DPASV responses toward 1.0 μM Pb2+. (B) Variation of Pb2+ peak current obtained from DPASV as a
function of release time. Error bars represent the standard deviation of three independent experiments.
1 mg·mL−1 Ab2 was added and was left to stand overnight at 4
°C, followed by adding 20 μL of the 1% BSA solution for 1 h
reaction. After centrifugation and washing thrice with a pH 7.4
phosphate buffer, the PbS CSs-Ab2 nanoprobe was finally
obtained and redispersed in 0.1 mL of pH 7.4 phosphate buffer
for further use.
Fabrication of Sandwich Immunosensor. Prior to each
experiment, GCE was sequentially polished with 0.3 and 0.05
μm of alumina powder, respectively. And the polished GCE
was successively sonicated with ethanol and distilled water to
remove any adsorbed substances on the electrode surface.
First, 6 μL of 1 mg·mL−1 C60-Chit dispersion was dropped
onto the electrode surface and dried at room temperature to
allow it to chemisorb onto the surface. The modified electrode
was washed with water and incubated with 6 μL of 5% GA for
2.5 h at 4 °C in refrigerator, followed by washing with
ultrapure water to remove unreacted reagents. Then, 6 μL of
10 μg·mL−1Ab1 was spread on the modified electrode at 4 °C
overnight, and excess antibodies were removed with PBS and
PBST, respectively. Afterward, 6 μL of 1% BSA solution was
added to the electrode surface and reacted for 1 h to block the
nonspecific sites. After another washing with PBS and PBST,
the immunosensor was obtained and stored at 4 °C prior to
use.
Measurement Procedure. To carry out the immuno-
reaction and electrochemical measurement, the immunosensor
was incubated at room temperature for 1 h with different
concentrations of HE4. After washing with PBS and PBST, the
immunosensor was further incubated with 6 μL of PbS CSs-
Ab2 nanoprobe for 45 min at room temperature. After finally
being washed with PBS to remove nonspecifically bounded
nanoprobes, the electrode was immersed into 200 μL of HNO3
solution (1 M) for 10 min to dissolve the probes. The resulting
solution was mixed with 4.8 mL of HAc-NaAc buffer (0.1 M,
pH 5.0) containing 0.2 mM Hg2+ for the detection of metal
ions by differential pulse anodic stripping voltammetry
(DPASV). The voltammogram was recorded in the potential
range from −1.2 to 0 V, with deposition potential of −1.0 V,
deposition time of 240 s, amplitude of 100 mV, pulse width of
50 ms, and quiet time of 2 s.
■ RESULTS AND DISCUSSION
Characterization of PbS CSs. To prepare the PbS CSs,
monodispersed PbS NPs with average diameter of 8 nm were
prepared and characterized (Supporting Information, Figure
2227
Article
S1). The CSs were synthesized through controlled agglomer-
ation of the PbS NPs at the droplet interfaces in the water-in-
oil emulsion, as illustrated in Figure 1A. To acquire cross-
linkable and stable PbS colloidosomes, the colloidosomes was
designed to be coated with polydopamine. Therefore, the
aqueous phase was chosen to be the pH 8.5 tris-HCl buffer
which was a proper medium for the self-polymerization of
dopamine.24 The organic phase was the mixture of vegetable
oil and heptane (V/V = 1/1), which is an excellent medium for
the dispersion of hydrophobic PbS NPs.
The morphology of the coarse products can be observed
clearly from the SEM image (Figure 1B) which exhibits
spherical particles with size of about 100 to 500 nm.
Magnifying one big particle (Figure 1C), the three-dimensional
spherical structures assembled by single nanoparticles can be
revealed. The hexagonally close packed surface, depicts more
closely in the further magnification (Figure 1D), emphasizes
the high order of the building block particles of the CSs. EDS
spectrum (Supporting Information, Figure S2) displays distinct
signal of Pb, S, C, and N, confirming the elemental
composition of the CSs. Therefore, nanosized PbS CSs were
prepared by inverse w/o Pickering emulsions containing oil-
soluble PbS NPs.
Since the coarse PbS CSs are not as homogeneous as
expected, the material was centrifuged, washed with ethanol
and redispersed in water. After that, the large CSs and the
surplus NPs were separated and PbS CSs with relatively
narrow size distribution were obtained. Furthermore, effect of
concentration of dopamine (CDA), loading amount of PbS NPs
(mPbS), oil-to-water volume ratio (Ro/w), and component of oil
phase on the formation of CSs was investigated, respectively.
Average size of CSs does not change much after addition of
low concentration of DA (Figure 2A,B). However, when CDA
increases from 0.5 to 2 mg·mL−1, a significant decrease in size
of CSs is observed (Figure 2C). The possible reason is that the
presence of high concentration of DA in the aqueous phase
influences the size of emulsion droplets. Moreover, when CDA
is held constant, an increase of mPbS from 5 to 20 mg lead to a
gradual increase in CSs size (Figure 2D−F), demonstrating
that more and more colloid particles are involved into the self-
assembly of PbS NPs onto the emulsion droplets. Since CSs
were usually prepared by self-assembly of colloid particles on
emulsion droplets, effect of Ro/w on the formation of the CSs
was investigated by changing volume of water. As shown in
Figure 2G−I, the average diameter of PbS CSs rarely changes
DOI: 10.1021/acs.analchem.8b04807
Anal. Chem. 2019, 91, 2224−2230
Analytical Chemistry
Figure 4. (A) DPASV responses toward blank (a, a′) and 100 pg·mL−1 HE4 (b, b′) by using PbS NPs (a, b) and PbS CSs (a′, b′) as labels. Inset is
the partial magnification for curve a and a′. (B) DPASV responses of the electrochemical immunoassay toward different concentration of HE4 and
the corresponding calibration curve (inset). Error bars represent the standard deviation of three independent experiments.
with increasing Ro/w values from 2 to 10, indicating that the
size of emulsion droplets is not influenced greatly by the
volume of water. However, the reduction of water volume
decreases the number of emulsion droplets which is depicted
by the density reduction of CSs on the SEM images. The size is
also tuned by changing the composition of the oil phase. The
reported oil phase for CSs preparation includes toluene,6
heptane,25 and vegetable oil.1,26 Considering the homogeneous
dispersion of PbS NPs, three types of organic phase were
studied. As shown in Figure 2J−L, comparing with toluene and
vegetable oil/toluene (1/1), vegetable oil/heptane (1/1) is an
ideal organic phase for the preparation of high-density and
homogeneous PbS CSs. In the subsequent experiments, PbS
CSs prepared with vegetable oil/heptane (1/1) as organic
phase and CDA = 1 mg·mL−1, Ro/w = 5, and mPbS = 10 mg were
applied. And the average diameter of CSs prepared at the
selected conditions, assessed via the analysis of dynamic
lighting scatter, is 112 nm (Supporting Information, Figure
S3).
Cargo Release of Pb2+. DPASV is an excellent technique
for the quantitative measurement of lead ions.22,27 By applying
a negative potential for predeposition and accumulation, a
well-defined peak owing to the electrochemical oxidation of Pb
can be clearly observed at −0.62 V on the voltammogram
(Figure 3A) recorded in pH 4.5 HAc-NaAc buffer containing
0.2 mM Hg2+. Owing to the excellent conductivity and
deposition capability of the C60-Chit nanocomposite,22 the Pb
oxidation peak can be dramatically enhanced by using the C60-
Chit modified GCE (C60-Chit/GCE; Figure 3A). Furthermore,
modification of the C60-Chit nanocomposite supplied an
excellent for the covalent bonding of the capture antibodies.28
With the aim of using the PbS CSs as the electroactive labels
for immunoassay, the acidity-triggered Pb2+ release from the
nanoassemblies was performed. Briefly, 200 μL of 1 M HNO3
solution was added into 6 μL of the suspension of the as-
prepared PbS CSs to trigger the release of Pb2+. And the
solution was diluted to 5.0 mL with HAc-NaAc buffer to
obtain a proper detection acidity of pH 4.5. Then, Pb2+ was
detected by DPASV using C60-Chit/GCE as working
electrode. Figure 3B shows the variation of the stripping
peak current with the addition time of HNO3, which ranged
from 1 to 30 min. The peak current increases with the increase
of the release time until 10 min. For release time longer than
10 min, the peak current increases slowly, showing the
approximation to complete release of lead ions within 10 min.
2228
Article
The concentration of the released Pb2+ from 6 μL of dispersion
of PbS CSs, evaluated using the quantitative relationship
between peak current on voltammogram and concentration
(Supporting Information, Figure S4A) is 7.9 μM, which is in
accordance with the result obtained from ICP-OES (Support-
ing Information, Figure S4B), demonstrating the “burst” of
lead ions.
PbS CSs-Based Electrochemical Immunoassay. In this
work, anti-HE4 antibodies (Ab1) were covalently cross-linked
onto the surface of C60-Chit/GCE to prepare the sensor
interface, and the secondary anti-HE4 antibodies (Ab2) were
immobilized onto the PbS CSs to introduce electroactive labels
for the electrochemical determination of HE4 based on the
formation of a sandwich-type immunocomplex. By sequentially
incubating with HE4 and the bioconjugates Ab2-PbS CSs,
DPASV response of the immunosensor was recorded. As
shown in Figure 4A, the response for blank solution (curve a′)
displays a small peak while for 100 pg·mL−1 HE4, a brilliant
oxidation peak with peak current of 46.13 μA (curve b′)
corresponding to the oxidation of lead was observed,
demonstrating the feasibility of the strategy. For comparison,
DPASV response of the same immuonsensing interface for 100
pg·mL−1 HE4 by using PbS NPs as electroactive nanoprobes
was also shown (curves a and b). The peak current for 100 pg·
mL−1 HE4 is only 0.64 μA which is about one-hundredth of
the peak current for PbS CSs labeling, indicating the dramatic
sensitivity difference of the two tracers for immunoassay.
DPASV response of the immunosensor toward different
concentrations of HE4 was recorded. As shown in Figure 4B,
the peak current increases with the increasing HE4 antigen
concentration. A good linear dependence between the current
and the logarithm of HE4 level can be achieved within the
dynamic working range from 10 fg·mL−1 and to 100 ng·mL−1
(inset of Figure 4B). The linear regression equation could be
fitted to be I (μA) = 10.51 × log C (pg·mL−1) + 26.65 (R2 =
0.9970). The DL is calculated to be 3.4 fg·mL−1, which is
about 2−4 orders of magnitude lower than the level of the
other reported methods (Table 1).
To demonstrate the sensitivity enhancement effect of PbS
CSs, we compared the stripping peak current of Pb2+ released
from the other lead-containing tracers for electrochemical
immunoassay (Table 2). Although the target for these assays is
different, we still can convince the relationship between the
stripping current and DL. Therefore, it can be concluded that
the large stripping peak current of Pb2+ was a decisive factor of
DOI: 10.1021/acs.analchem.8b04807
Anal. Chem. 2019, 91, 2224−2230
Analytical Chemistry Article

Table 1. Comparison of Analytical Characteristics for HE4 stability of the immunosensor was also evaluated by storing the
Assay immunosensor and the probes at 4 °C for 2 weeks. The
electrochemical response only decreased 3.3% for HE4
detection
method linear range limita ref compared with the freshly prepared, which indicated the
proposed immunosensor has good stability.
photoelectrochemical 0.025−4 ng·mL−1 15.4 pg·mL−1 29
sensor Application in Analysis of Serum Samples. To
electronic sensor 1.5−25 ng·mL−1 100 pg·mL−1 30 corroborate the analytical accuracy of the new developed
LSPR biosensor 10−10000 pM 4 pM 31 electrochemical immunoassay by using PbS CSs-Ab2 as
chemiluminescent 20−1500 pM 0.18 pM 32 electrochemical labels to detect HE4 in real samples, serum
immunoassay samples diluted with PBS buffer (1:10) were spiked with
ELISA 15−900 pM 15 pM 33 different concentrations of HE4. The recoveries of the spiked
ELISA 4−400 pM 6.8 fM 34 HE4 ranged from 95.5% to 111.8% (Supporting Information,
electrochemical 3−300 pM 0.06 pM 35 Table S1), indicating great accuracy and reliability of the
immunoassay proposed great accuracy and reliability of the proposed
electrochemical 20−40000 pM 12 pM 23
immunoassay immunosensor the quantification of HE4 in biological samples.
electrochemical

a
immunoassay
0.00001−100
ng·mL−1
Molecular weight of HE4 is about 25 kDa.
3.4 fg·mL−1

34
this work
the high sensitivity of the ultimate electrochemical immuno-
assay. In this work, even for the low concentration of 10 fg·
mL−1 HE4, the related Pb2+, determined by ICP-OES, was 3.7
μM. Therefore, the mechanism for the ultrahigh sensitivity of
the PbS CSs-based immunoassay was attributed to the
following three aspects. First, the cascade Pb2+ releasing
capability of the PbS CSs exceptionally amplified the
antibody−antigen interaction. Second, the unique ability to
preconcentrate target metal ions and the inherent detection
sensitivity of DPASV ensured the intensity of the electro-
chemical signal of the released Pb2+. Third, the signal of the
released Pb2+ was further amplified a hundredfold by the
application of C60-Chit nanocomposite.
Specificity, Reproducibility, and Stability of the
Immunoassay. The response of the immunoassay for several
biomolecules that might coexist with HE4 in the real serum
samples were detected. Compared with the current response
obtained for 100 pg·mL−1 HE4, the variation in current caused
by the mixture of HE4 and equal concentration of
■ CONCLUSIONS
In summary, we have presented a novel method to prepare
nanometer-sized (∼112 nm) PbS CSs from inverse w/o
Pickering emulsion containing PbS NPs (∼8 nm) and devised
a colloidosome-based electrochemical immunosensing plat-
form for the ultrasensitive detection of the ovarian cancer
biomarker HE4. Introduction of C60-Chit in this system was
conducive to amplify the detection sensitivity to Pb2+ during
the measurement, whereas utilization of PbS CSs was dramatic
to enhance the released amount of Pb2+ signal tags. The
developed immunoassay exhibited an ultralow detection limit
of 3.4 fg·mL−1 and potential application in patient serum
sample analysis. In addition, this system also provides a new
technological platform for the study of other biomarkers.
Moreover, the voltammetric signal can be changed by various
CSs (e.g., CuS and CdS) and even to offer a highly sensitive
and selective simultaneous bioelectronic detection of several
protein targets to meet the requirements of specific
applications.
■
*
ASSOCIATED CONTENT
S Supporting Information

carcinoembryonic antigen (CEA), immunoglobulin G (IgG),
human serum albumin (HSA), or excessive level of glucose
(Glu) was less than 5% (Supporting Information, Figure S5A),
indicating the satisfying specificity of the developed method
toward HE4. To investigate the reproducibility of the
developed assay, a batch of five parallel experiments was
conducted under the same condition, where 1 pg·mL−1 of HE4
was detected (Supporting Information, Figure S5B). As a
result, a relative standard deviation of 5.9% was achieved,
indicating good reproducibility of the developed method. The
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.8b04807.
Synthesis of oleylamine-capped colloidal PbS NPs.
Preparation of MPA-capped water-soluble PbS NPs.
Figure S1, SEM, TEM images, and XRD pattern of the
as-synthesized PbS NPs. Figure S2, EDS spectrum of
PbS CSs. Figure S3, DLS intensity distribution of PbS
CSs. Figure S4, Quantitative determination of Pb2+.
Figure S5, Electrochemical responses of the developed

Table 2. Comparison of Electrochemical Immunoassay Using Pb2+-Based Labels

labelsa targetb peak current of Pb2+c detection limit(pg·mL−1) ref
−1
PbS CSs HE4 45.76 μA for 0.1 ng·mL HE4 0.0034 this work
PbS@PDA IgM 0.1 μA for 0.1 ng·mL−1 of IgM 1.3 × 107 36
AuNPs-Ab2-Pb2+ AFP 9.1 μA for 0.1 ng·mL−1 AFP 3.1 37
rApo-Pb CEA 11.5 μA for 0.1 ng·mL−1 CEA 0.35 38
Au/BSA-Pb2+ CEA 14.2 μA for 0.1 ng·mL−1 CEA 0.08 39
PbS QDs lysozyme 0.7 μA for 1 ng·mL−1 lysozyme 40
PbS QDs BSA 1.5 μA for 50 ng·mL−1 BSA 0.01 18

a
PbS@PDA: polydopamine particles loaded with PbS quantum dots. rApo-Pb: recombinant apoferritin-encoded Pb nanoparticles. QDs: quantum
dots. bIgM: immunoglobulins M. AFP: α-fetoprotein. CEA: carcinoembryonic antigen. cPeak currents for all references are calculated from the
according relationship between peak current and concentration of target.

2229 DOI: 10.1021/acs.analchem.8b04807

Anal. Chem. 2019, 91, 2224−2230
Analytical Chemistry Article

immunoassay. Table S1, Detection of HE4 in spiked (24) Lai, G.; Zheng, M.; Hu, W.; Yu, A. Biosens. Bioelectron. 2017,
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■ AUTHOR INFORMATION
Corresponding Authors
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Gordon, V. D.; Chen, X.; Hutchinson, J. W.; Weitz, D. A. Langmuir
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*E-mail: zhengjb@nwu.edu.cn Interfaces 2015, 7, 15935−15943.
ORCID (28) Zhang, H.; Ma, L.; Li, P.; Zheng, J. Biosens. Bioelectron. 2016,
Hongfang Zhang: 0000-0002-3255-1857 85, 343−350.
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■ ACKNOWLEDGMENTS
This work is financially supported by National Natural Science
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2230 DOI: 10.1021/acs.analchem.8b04807

Anal. Chem. 2019, 91, 2224−2230