ARTICLE

pubs.acs.org/ac

Highly Sensitive Immunoassay Based on Immunogold-Silver

Amplification and Inductively Coupled Plasma Mass Spectrometric
Detection
Rui Liu,† Xing Liu,† Yurong Tang,† Li Wu,‡ Xiandeng Hou,†,‡ and Yi Lv*,†
†
College of Chemistry and ‡Analytical and Testing Center, Sichuan University, Chengdu, Sichuan 610064, China

ABSTRACT: In this work, we demonstrated a highly sensitive induc-

tively coupled plasma mass spectrometric (ICPMS) method for the
determination of human carcinoembryonic antigen (CEA), which com-
bined the inherent high sensitivity of elemental mass spectrometric
measurement with the signal amplification of catalytic silver deposition
on immunogold tags. The silver amplification procedure was easy to
handle and required cheap reagents, and the sensitivity was greatly
enhanced to 60-fold after a 15 min silver amplification procedure. The
experimental conditions, including detection of gold and silver by
ICPMS, immunoassay parameters, silver amplification parameters, analytical performance, and clinical serum samples analysis,
were investigated. The ICPMS Ag signal intensity depends linearly on the logarithm of the concentration of human CEA over the
range of 0.07-1000 ng mL-1 with a limit of detection (LOD, 3σ) of 0.03 ng mL-1 (i.e., 0.15 pM). The LOD of the proposed
method is around 2 orders of magnitude lower than that by the widely used enzyme-linked immunosorbent assay (ELISA) and
1 order of magnitude lower than that by clinical routine chemiluminescence immunoassay (CLIA) or time-resolved
fluoroimmunoassay (TRFIA) and conventional ICPMS immunoassay. The present strategy was applied to the determination
of human CEA in clinical human serum samples, and the results were in good agreement with those obtained by
chemiluminescence immunoassay.

A highly sensitive technique for protein quantification plays an

important role in the early diagnosis and elucidation of
molecular mechanisms for many diseases, because even a few
molecules of proteins are sufficient to affect the biological func-
tions of cells and trigger pathophysiological processes.1 Unfortu-
nately, in many cases, it is still hard to use conventional methods
to detect some important protein biomarkers, due to their low
abundance in body fluids or tissues. Hence, it is extremely urgent
to develop highly sensitive methods for detection of low abun-
dant proteins. Generally, high sensitivity can often be obtained
using a signal amplification procedure. Many signal amplification
schemes have been reported for sensitive bioanalyte quantifica-
tion, such as rolling circle amplification,2-5 avidin-biotin ampli-
fication,6,7 inline atom transfer radical polymerization,8 and
exponential isothermal amplification.9 However, they usually
require rather complex reagents or sophisticate protocols.
Due to its easy handling, cheap reagents, robustness, and high
sensitivity,10,11 the immunogold-silver amplification technique
has been widely used and even successfully commercialized for
the past 30 years since it was first developed in 1983 by Holgate
et al.12 and Danscher et al.13 It is initially developed and com-
monly applied as a sensitive and specific immunohistochemical
visualization technique.10 Later, Mirkin and co-workers devel-
oped a scanometric DNA array,14 an electrical detection-based
DNA array,15 and a Raman spectroscopic fingerprints scheme for
DNA and RNA detection16 using this technique for sensitivity
improvement. Immunogold-silver amplification has also been
explored for bioanalyte quantification by coupling to microgravi-
metric biosensor,17 electrochemical stripping analysis,18,19 chemi-
luminescent analysis,20 dot-blot immunoassay,21,22 and micro-
fluidic chips.23,24
Inductively coupled plasma mass spectrometry (ICPMS) is
unarguably the predominant and most sensitive commercial
instrument for the determination of a wide range of metals and
several nonmetals. The advantages of ICPMS include low detec-
tion limits, low matrix effects, large dynamic ranges, and high
spectral resolution for elements and isotopes.25,26 Therefore,
element tagged immunoassay with ICPMS detection has become
an emerging technique in immunoassay research27-30 since it
was first reported by Zhang et al.31,32 ICPMS as a readout tool
does not require nanoparticle reporters to possess the optical,
electric, electrochemical, magnetic, or any other special proper-
ties since atomic ions from the nanoparticle are directly detected.
A distinguished feature of this method is the great multiplexing
potential for biological analytes endowed by the excellent
element isotopic spectral resolution of the mass spectrometer.33-36
Another feature is that high sensitivity could be easily obtained by
the use of the nanoparticle tag instead of metal ions, due to large
quantities of detectable atoms in each nanoparticle tag.32,37
Methods for the detection of biological analytes such as small
Received: December 15, 2010
Accepted: February 2, 2011
Published: February 24, 2011

r 2011 American Chemical Society 2330 dx.doi.org/10.1021/ac103265z | Anal. Chem. 2011, 83, 2330–2336
Analytical Chemistry ARTICLE

Table 1. Working Parameters of ICPMS

parameters values

RF power/W 1200
cool gas flow/L min-1 13
auxiliary gas flow/L min-1 0.8
nebulizer gas flow/L min-1 0.85
sample uptake rate/mL min-1 1
torch shield torch
resolution standard
dwell time/ms 10
channels 3
sweeps 100
38 31,32,34,35,37,39-44
biomolecules, proteins, nucleic acids,45,46 and
47,48
even cells have been proved to be successful. Additionally, it
is believed to be the ideal technique for the next generation of
flow cytometers.47,49 It should be mentioned that Mcleod et al.
combined immunohistochemistry with laser ablation ICPMS
using the gold nanoparticles (Au-NPs) label and subsequent silver
amplification for imaging of breast cancer-associated proteins.50
The method showed higher sensitivity and good resolution as
compared to optical microscopy. Apparently, it is worthwhile to
further explore quantitative application after silver amplification.
In this work, therefore, a highly sensitive immunogold-silver
amplified ICPMS immunoassay is presented through the
catalytic precipitation of silver on the immunogold nanopar-
ticles, which combined the intrinsically high sensitivity of
ICPMS with the signal enhancement of immunogold-silver Figure 1. TEM images of colloidal gold before (a) and after (b) 15 min
amplification. Carcinoembryonic antigen (CEA), a glycopro- silver amplification.
tein of 200 kDa that has been extensively studied as a tumor
(Au-NPs) and secondary CEA antibody-colloidal Au conjugates
marker for clinical diagnosis and the individual’s annual
were synthesized in our laboratory. Unless otherwise stated, all
medical checkup, was chosen as the model analyte. It is used
the reagents used in this study were at least of analytical grade
as part of a panel of cancer markers for different cancers and
and obtained from Changzheng Chemical Reagent Co. Ltd.
even an independent prognostic factor, since it is present in
(Chengdu, China).
about 95% of all colon tumors and 50% of breast tumors and is
The buffers used were as follows: (A) coating buffer, 0.05 M
also associated with ovarian carcinoma, lung cancer, and other
carbonate/bicarbonate buffer solution, pH 9.6 (dissolve 2.601 g
cancers.1
of Na2CO3 and 3.437 g of NaHCO3 in 1 L of deionized water);
(B) blocking buffer, 1% (w/v, g mL-1) BSA in 0.01 M sodium
’ EXPERIMENTAL SECTION phosphate buffered saline (PBS, dissolve 2.204 g of Na2H-
PO4 3 12H2O, 0.600 g of NaH2PO4 3 2H2O, and 8.766 g of
Instrumentation. An X Series ICPMS (Thermo Electron Co., NaCl in 1 L deionized water), pH 7.4. The blocking buffer was
Winsford, Cheshire, UK) was used throughout this work. A stored at 4 °C and used within a week; (C) assay buffer, 0.01 M
standard glass concentric nebulizer and a standard glass conical PBS containing 1% BSA (w/v), pH 7.4; (D) washing buffer,
impact bead spray chamber were employed with an uptake rate of 0.01 M PBS with 0.05% (v/v) Tween 20, pH 7.4; and (E)
around 1 mL min-1. Single-channel and multichannel (8-channel) citrate buffer, pH 3.5 (dissolve 23.5 g of trisodium citrate
pipettes (Finnpipette Color, Thermo Scientific, USA) were used dihydrate and 25.5 g of citric acid monohydrate in 850 mL of
for convenient and rapid transfer of the solutions. Prior to deionized water). This buffer can be kept at 4 °C for at least 2
analysis, the instrumental parameters were tuned using a 1% to 3 weeks. Before use, it is adjusted to pH 3.8 with citric acid
HNO3 solution containing 10 ng mL-1 In and Pb. The opti- solution.
mized parameters are listed in Table 1. The morphologies of the Silver amplification solutions (A and B) are prepared freshly:
samples were observed with a transmission electron microscope Solution A, dissolve 80 mg of silver acetate in 40 mL of deionized
(TEM, JEM-100CXII). water (silver acetate crystals can be dissolved by continuous
Reagents and Immunoreaction Buffers. Deionized water stirring within about 15 min) and solution B, dissolve 200 mg
with conductivity of 18.2 MΩ cm-1 from a water purification of hydroquinone in 40 mL of citrate buffer. Solution A and
system (ULUPURE, Chengdu, China) was used in this work. solution B were mixed immediately with an equal volume
Polystyrene 96-well microtiter plates (468667, NUNC, Denmark) before use.
were used to perform the immunoreactions. CEA antigen, primary Preparation of Colloidal Gold Nanoparticles. Briefly, after
CEA antibody, secondary CEA antibody, and bovine serum boiling 100 mL of 0.01% (m/v) HAuCl4 3 4H2O with 4 mL of 1%
albumin (BSA) were purchased from Bejing Biosynthesis Bio- (m/v) trisodium citrate in aqueous solution for 30 min, the
thechnology Co. (Beijing, China). Colloidal gold nanoparticles resultant colloidal suspension was cooled and stored at 4 °C. The
2331 dx.doi.org/10.1021/ac103265z |Anal. Chem. 2011, 83, 2330–2336
Analytical Chemistry
Figure 2. Schematic diagram of the sandwich immunoassay and silver
amplification for human CEA with Au-NP tags.
average diameters of Au-NPs were 12 nm, as confirmed by TEM
(Figure 1a).
Preparation of Colloidal Gold-Antibody Conjugates. The
Au-NP suspension was subjected to 5 min ultrasonication
before conjugation. The secondary CEA antibody (10% more
than the minimum amount, which was determined using a
flocculation test) was added to 1 mL of pH-adjusted colloidal
Au suspension followed by incubation at room temperature
for 10 min. The conjugates were centrifuged at 14 000 rpm for
10 min, and the soft sediment was resuspended in 0.01 M PBS.
A 1 mL suspension of Au-NPs was tagged with 40 μg of
secondary CEA antibody. Addition of glycerol to a final concen-
tration of 50% and BSA to a final concentration of 1% allows the
storage of the colloidal Au-antibody conjugates at -20 °C for
several months.
Immunoassay Principles. The assay was performed in a
polystyrene 96-well microtiter plate. The schematic diagram of
the sandwich immunoassay and silver amplification for human
CEA with Au-NP tags was shown in Figure 2. Initially, 200 μL of
10 μg mL-1 primary CEA antibodies in coating buffer was
steadily attached on the solid polystyrene substrate via physical
adsorption between hydrophobic groups of antibody molecule
and polystyrene (step 1). The glass slide was washed off three
times with 350 μL of washing buffer to remove any unbound
antibodies, and the uncoated active sites of polystyrene substrate
were saturated with 300 μL of blocking buffer, in which BSA was
used as a blocking agent to prevent nonspecific adsorption of the
antigens in the next step. BSA was also added to the antigen and
Au-NPs conjugates solutions to minimize nonspecific binding
and aggregation. Diluted CEA standard solutions (in assay buffer)
or serum samples of 100 μL and diluted secondary CEA
antibodies-colloidal gold conjugates of 100 μL were incubated
over the polystyrene substrate to allow the formation of sandwich
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ARTICLE
Figure 3. Calibration curves for Ag and Au by ICPMS.
complexes in the microtiter plate (step 2). Unbound antigens
and gold conjugates were removed from the glass slide with
350 μL of washing buffer (three times) and 400 μL of pure water
(three times). The attached Au-NPs were enlarged by 200 μL of
silver amplification A and B mixture solution for silver metal
deposition (step 3). Silver amplification A and B mixture solution
was rapidly transferred to microtiter plates by use of a multi-
channel pipet, and a stopwatch was used to control exact reaction
time. Silver amplification proceeds as an autocatalytic reaction:
the Au-NPs serve as nucleation sites to catalyze the reduction of
silver ions to metallic silver. Immediately after a 15 min ampli-
fication, the reaction was stopped by pouring out all the solutions
from the microtiter plate. The metallic silver on the enhanced
plates were washed with 400 μL of pure water (three times),
dried, dissolved with 200 μL of 50% (v/v) high purity nitric acid
solution for 10 min, and diluted to 4 mL with pure water. The
atomic mass spectrometric signals obtained from the diluted
solution were recorded by ICPMS for analyte quantification
(step 4).
’ RESULTS AND DISCUSSION
The present work aims at developing a highly sensitive
ICPMS immunoassay by detecting the silver deposited on the
Au-NPs. The proposed silver amplified immunogold ICPMS
immunoassay combines the inherent high sensitivity of
ICPMS detection with the signal amplification resulting from
the catalytic precipitation of silver on the Au-NPs tags,
pushing the sensitivity of the immunoassay to the low pico-
molar domain. The analytical procedure consists of the
immunoreaction of the antigen with the primary antibody
adsorbed on the walls of a polystyrene microtiter plate and a
secondary colloidal gold-labeled antibody to form a sandwich
complex, silver amplification, acid dissolution, and ICPMS
detection of the silver (Figure 2). ICPMS was used earlier for
determining the immunogold tags after immunoassay; thus,
the comparison of analytical performance before and after
silver amplification was also investigated. The detailed opti-
mization and performance characteristics of the developed
silver amplified ICPMS immunoassay are reported in the
following sections.
Detection of Gold and Silver by ICPMS. Au and Ag were
detected using the 197Auþ and 107Agþ signal whose isotopic
abundances are 100% and 51.35%, respectively. Theoretically,
high interferences may be produced from 197TaOþ (99.7%)
and 107 NbNþ (99.6%) to 197Auþ and 107Agþ. However, under
the operating conditions of the proposed method, Ta and Nb
atoms can be controlled at very low concentration levels using
dx.doi.org/10.1021/ac103265z |Anal. Chem. 2011, 83, 2330–2336
Analytical Chemistry
Figure 4. Dependence of the ICPMS Ag signal upon immunoassay
parameters. (a) The antigen-antibody immunoreaction time. Concen-
tration of human CEA, 1 μg mL-1; dilution ratio of the antibody-
colloidal gold conjugate, 1:20; and silver amplification time, 15 min.
(b) The dilution ratio of antibody-colloidal gold conjugate. Concentration
of human CEA, 1 μg mL-1; antigen-antibody immunoreaction time,
60 min; and silver amplification time, 15 min.
Figure 5. Dependence of the ICPMS signal upon silver sources and
amplification time. Concentration of human CEA, 10 ng mL-1; anti-
gen-antibody immunoreaction time, 60 min; and dilution ratio of the
antibody-colloidal gold conjugate, 1:20.
high purity reagents and deionized water. Therefore, the effects
of 197TaOþ and 107NbNþ overlapping the 197Auþ and 107Agþ
signals are negligible. Both 197Auþ and 107Agþ are suitable for
our application. The optimizations of ICPMS parameters were
carried out to obtain the maximum sensitivity. Parameters such as
radio frequency (rf) power, cool gas flow rate, and nebulizer gas
flow in ICPMS were optimized and listed in Table 1.
Under the optimum conditions, the ICPMS calibration curves
for Au and Ag were constructed with a serial of elemental standard
solutions (Figure 3). The limits of detection (LOD, 3σ) for Au
and Ag elements were measured as 0.001 and 0.002 ng mL-1,
2333
ARTICLE
respectively. Linear ranges of more than 5 orders of magnitude
were routinely obtained, with R2 > 0.999.
Optimization of Immunoassay Conditions. The proposed
ICPMS immunoassay of human CEA was performed as depicted
in Figure 2. Immunoassay parameters including immunoreac-
tion time and the dilution rate of antibody-immunogold con-
jugates were optimized using 1 μg mL-1 CEA solution. The
dependence of the antigen-antibody reaction time upon the
Ag signal was shown in Figure 4a. The response increased
rapidly with the reaction time between 20 and 60 min and then
leveled off above 60 min. This indicates that the interaction of
antigen with antibody has reached equilibrium after 60 min, and
hence, a reaction time of 60 min was selected for further
investigations.
During the colloidal gold conjugate-based immunoassay, the
dilution rate of antibody-colloidal gold conjugates is a key factor
affecting the detection sensitivity of the method, as was system-
atically studied in a previous report.51 In our study, the influence
of the concentration of the antibody-colloidal gold conjugates
upon the response of the ICPMS Ag signal was surveyed in the
range of 1:10 to 1:200 dilutions. As shown in Figure 4b, the
ICPMS Ag signal intensity remained almost a constant value
while increasing the dilution rate (i.e., decreasing the con-
centration) of the colloidal gold-labeled antibody from 1:10 to
1:20 and then decreased in a nearly linear fashion by increasing
the dilution rate. A dilution ratio of 1:20 was consequently selected
for further studies.
Silver Amplification. Immunogold silver amplification has
been extensively studied and widely used in histochemical micro-
scopy studies for 30 years,10 where functional immunogold acts
as a catalyst to reduce silver ions to metallic silver in the presence
of a reducing agent (such as hydroquinone). The autometallo-
graphic silver deposition procedure enlarges the size and darkens
the color of the gold particles, such that protein-, antibody-, or
DNA-conjugated Au-NPs become visible under electron or
optic microscopy. In the subsequent quantitative applications of
this technique, the sensitivity of detection in the case of Au-NP
probes can be increased drastically by up to 5 orders of
magnitude.11,52 The ingredient of silver amplification solution
was adopted from Danscher et al.13,53 and Hacker et al.54
Apparently, when the molar concentration of each component
of the silver amplification solution is fixed, the performance of
silver amplification by catalytic precipitation on the Au-NPs tags
would be strongly influenced by the silver source and silver
amplification time.
Silver Source. A number of silver salts, i.e., silver nitrate,19,20
silver lactate,13,53 and silver acetate,54,55 have been used as the
silver amplification sources. The silver nitrate and silver lactate
were light sensitive, and the amplification procedure conse-
quently needed to be carried out in darkroom conditions or in
a dark cupboard, while silver acetate was considered to be light
insensitive during silver amplification. We have studied the per-
formance of silver nitrate and silver acetate as silver sources, since
they are widely used and commercially available. The silver
amplifications by silver nitrate and silver acetate were compared
in a dark room (Figure 5). As shown in Figure 5, a comparable
performance of these two silver sources was obtained. However,
silver amplification by silver nitrate gave rather a low signal-to-
noise ratio under the light, while the performance of silver acetate
remained almost invariable, which was in good accordance with
the previous research.54 The light-insensitive silver acetate source
was selected for the further study for the convenience of operation.
dx.doi.org/10.1021/ac103265z |Anal. Chem. 2011, 83, 2330–2336
Analytical Chemistry
Figure 6. Relationship between the concentration of CEA and visible or
ICPMS signal. (a) The photograph of microtiter plate after immunogold
labeled sandwich immunoassay; (b) The photograph of microtiter plate
after immunogold labeled sandwich immunoassay and silver amplifica-
tion; and (c) The ICPMS Ag and Au signal after dissolving the metallic
elements in the microtiter plate.
Figure 7. Specificity for the determination of human CEA using the
proposed immunoassay. Concentration of human CEA, 5 ng mL-1; and
concentration of the other proteins, 100 ng mL-1.
Silver Amplification Time. We studied the appropriate time
duration of silver amplification. As shown in Figure 5, the
ICPMS signal of deposited silver increased nearly linearly with
the silver amplification time. However, longer silver amplifi-
cation time, while offering very favorable signal enhancement,
led to an increase in the background. In contrast to the
analytical signal generated by the silver deposited exclusively
on the Au-NPs tags, such a background response might result
from nonspecific binding of silver ions onto the walls of the
polystyrene microtiter plate or the immobilized proteins,
2334
ARTICLE
which also increases with the silver amplification time. Con-
sidering both signal sensitivity and background noise, we
chose a 15 min silver amplification time for the further
experiments. Figure 1 displays TEM images of colloidal gold before
(a) and after (b) silver amplification. It can be seen that the
diameters of the 12 nm Au-NPs increased dramatically to over
100 nm after 15 min of silver deposition/amplification.
Analytical Performance. Under these optimal conditions
described above, a negligible red color of the antibody-colloidal
gold conjugate can be observed on the walls of a polystyrene
microtiter plate after sandwich immunoassay (Figure 6a), which
indicated that the immunogold labeled antibody has been adsorbed
on the walls of the polystyrene microtiter plate. After 15 min
of silver amplification, a naked-eye visible black color evi-
dently appeared (Figure 6b), which visually confirmed the
formation of metallic silver shell over Au-NPs labels. As low as
10 ng mL-1 CEA can be directly visualized, demonstrating a
potential application for visual detection. Subsequently, the
relationship between the concentration of CEA and ICPMS
signal was investigated. As shown in Figure 6c, the dynamic
range of the CEA concentration from 0.07 to 1000 ng mL-1
was obtained by the use of Ag ICPMS signal. The correlation
equation was Y = 8.3  105 lg[CEA] þ 1.4  106, with the
correlation coefficient r = 0.9917. The sensitivity enhance-
ment of Ag over Au signal was up to 60-fold, i.e., the ratio of
the slopes of the two calibration curves using the Ag and Au
ICPMS signal, and the limit of detection (LOD, 3σ) was 0.6
and 0.03 ng mL-1 before and after silver amplification
(calculated with Au and Ag signal intensity), respectively.
The deviation from linearity was observed when the concen-
tration of CEA was higher than 1000 ng mL-1. The reprodu-
cibility expressed as relative standard deviation (RSD) of
10 ng mL-1 CEA was 3.8% for within-batch (intra-assay) or
5.3% for between-batch (inter-assay).
The specific recognition of antigenic species for the proposed
ICPMS immunoassay was also investigated, with results shown
in Figure 7. It can be seen that only the CEA can be recognized in
the sandwich-type immunoreaction. Human IgG, rabbit IgG,
goat IgG, or human AFP does not significantly interfere with the
determination of CEA.
A comparison of analytical performance of the present method
with those of the other widely used immunoassay methods for
the determination of CEA is given in Table 2. The LOD of the
present method has more than 1 order of magnitude improve-
ment compared to that of conventional Au-NP labeled ICPMS
immunoassays and is even better than Eu3þ labeled laser ablation
ICPMS immunoassay.35 It is also around 2 orders of magnitude
lower than that of the widely used enzyme-linked immunosor-
bent assay (ELISA) or 1 order of magnitude lower than the
clinical routine chemiluminescence immunoassay (CLIA) and
time-resolved fluoroimmunoassay (TRFIA) for the determina-
tion of human CEA.
Clinical Serum Sample Analysis. Sensitive detection of CEA
is essential in clinical laboratories because increased correlation
levels are found with a number of cancers including early color-
ectal, lung, breast, pancreatic, and bladder cancers.1 Clinical human
serum samples were donated by Chengdu 7th People’s Hospital
(Chengdu, China). As shown in Table 3, analytical results of the
proposed method agree well with those by clinical chemilumi-
nescent (CL) immunoassay, indicating that the present method
could be applied to real clinical samples.
dx.doi.org/10.1021/ac103265z |Anal. Chem. 2011, 83, 2330–2336
Analytical Chemistry ARTICLE

Table 2. Comparison of Various Immunoassay Methods for CEA Determinationa

tags immunoassay format analytical method limits of detection/ng mL-1 references

Au microwells/sandwich-type ICPMS immunoassay after silver amplification 0.03 (0.15 pM) this work
Au microwells/sandwich-type ICPMS immunoassay 0.6 (3 pM) this work
Eu3þ microwells/sandwich-type laser ablation ICPMS immunoassay 0.14 35
HRP microwells/sandwich-type enzyme-linked immunosorbent assay 2 43
BHHCT-Sm3þ microwells/sandwich-type time-resolved fluoroimmunoassay 0.30 56
BPTA-Tb3þ microwells/sandwich-type time-resolved fluoroimmunoassay 0.20 57
HRP microwells/sandwich-type chemiluminescence immunoassay 0.61 58
DMDSBA microwells/sandwich-type chemiluminescence immunoassay 0.53 59
a
Au, Au-NPs; BHHCT-Sm3þ, Sm3þ chelate of 4,40 -bis (100 ,100 ,100 ,200 ,200 ,300 ,300 -heptafluoro-400 ,600 -exanedion-600 -yl)-chlorosulfo-o-terphenyl; BPTA-
Tb3þ, Tb3þ chelate of N,N,N1,N1-(2,6-bis(30 -aminomethyl-10 -pyrazolyl)-4-phenyl pyridine) tetrakis (acetic acid); HRP, horseradish peroxidase;
DMDSBA, 10,100 -dimethyl-3,30 -disulfo-9,90 -biacridine.

Table 3. Analytical Results of Human Serum Samples ’ REFERENCES

sample a
this method /ng mL -1 b
CL /ng mL
human serum 1 1.2 ( 0.1 1.4
human serum 2 2.5 ( 0.3 2.7
human serum 3 3.4 ( 0.4 3.5
human serum 4 35.8 ( 2.0 35.6
human serum 5 159.5 ( 10.3 167.7
a
Obtained value ( standard deviation. b CL, chemiluminescence im-
munoassay was performed by Chengdu 7th People’s Hospital.
’ CONCLUSIONS
The feasibility of a highly sensitive ICPMS immunoassay based
on the quantitative precipitation of silver onto immunogold tags
has been demonstrated. The great signal enhancement by silver
amplification is successfully combined with the sensitive ICPMS
detection. The silver amplification procedure is simple, low cost,
and rapid. The proposed method is competitive to some con-
ventional methods such as ELISA, CLIA, and TRFIA in clinical
analysis for CEA, which also showed great potential for numer-
ous applications in sensitive immunoassay, DNA hybridization,
and other biological analysis. Other signal amplification schemes,
such as rolling circle amplification, avidin-biotin amplification,
and exponential isothermal amplification, may also exhibit some
intriguing advantages for biosample analysis by coupling the
sensitive ICPMS immunoassay. Furthermore, the ICPMS im-
munoassay has a wide choice of metal nanoparticle labels and the
multielement detection power; thus, highly sensitive simulta-
neously multiplex immunoassay may be realized by incorpora-
tion of different signal amplification methods.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: lvy@scu.edu.cn. Tel./Fax: þ86-28-8541-2798.
’ ACKNOWLEDGMENT
Authors acknowledge the financial support for this project
from the National Natural Science Foundation of China (Nos.
20835003 and 21075084) and the Sichuan Youth Science and
Technology Foundation (No. 2009-18-409). Mr. Hao Lv from
Chinese Academy of Measurement and Testing Technology is
thanked for helping with handling ICPMS. The authors also
thank Mr. Guanglei Cheng of the Analytical and Testing Center
for his assistance with analytical atomic spectrometric measurements.
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