DOI: 10.1002/chem.

200801722

Gold Nanolabels for New Enhanced Chemiluminescence Immunoassay of

Alpha-Fetoprotein Based on Magnetic Beads
Sai Bi, Yameng Yan, Xiaoyan Yang, and Shusheng Zhang*[a]
Abstract: A novel and sensitive chemiluminescence immunoassay (CLIA)
has been developed by employing a
new chemiluminescence (CL) enhancer, bromophenol blue (BPB), for the
determination of alpha-fetoprotein
(AFP) based on magnetic beads (MBs)
and colloidal gold nanoparticles
(AuNPs) modified with HRP-labeled
anti-AFP antibodies. BPB, as a chemical indicator, was found to act as a
novel and highly signal enhancer of the
peroxidase-catalyzed CL reaction of lu-

minol with hydrogen peroxide. After

optimizing the CL reaction conditions,
this new luminolH2O2HRPBPB CL
system was applied to a sandwich-type
CLIA based on the magnetic separation and the amplification feature of
AuNPs as HRP labels. A linear range
was obtained when the concentrations
Keywords: gold immunoassays
luminescence magnetic beads
nanoparticles

Introduction
Since radioimmunoassays (RIA) were introduced in the
early 1960s, they have played a major role in medicine and
related areas due to their high sensitivity and specificity.[1]
However, this method involves radioactive materials, sophisticated instrumentation and is time and sample consuming.[2]
Therefore, enzyme immunoassays (EIA) have become an
important analytical method in clinical diagnoses.[35] The
improvement of EIA in terms of reducing assay time and
simplifying operations is one of the major trends in development of immunoassay technology.[6]
In recent years, many improvements have been made by
using magnetic beads (MBs) and gold nanoparticles
(AuNPs) in order to enhance sensitivity and reduce detection time.[7] Magnetic beads (MBs), as a special biomolecu[a] Dr. S. Bi, Y. Yan, Dr. X. Yang, Prof. S. Zhang
Key Laboratory of Eco-chemical Engineering
Ministry of Education
College of Chemistry and Molecular Engineering
Qingdao University of Science and Technology
Qingdao 266042 (China)
Fax: (+ 86) 532-8402-2750
E-mail: shushzhang@126.com
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200801722.

4704

of AFP were from 0.1 to 5.0 ng mL 1

(R = 0.9997) with the detection limit of
0.01 ng mL 1 (3s), which is one order
of magnitude lower than that obtained
without using AuNPs, and much lower
than that typically achieved by ELISA.
The present method was successfully
applied to the determination of AFP in
human serum samples. The results indicated that this proposed protocol could
be quite promising for the application
in immunoassays.

lar immobilizing carrier, have been widely applied in immunoassays; enzyme, DNA and protein immobilization and purification; and magnetically controlled transport of anticancer drugs.[811] An analytical method using MBs has many advantages in that they have potentially larger surface area
which may provide a high density of antibody immobilization, and target antigen in clinical or human serum samples
can be directly captured, easily separated and even concentrated by antibody-coated MBs, and analyzed by immunoassay.[1215] Up to now, several novel ultrasensitive detections
of proteins have been developed and used in clinical diagnoses based on MBs.[1618]
Colloidal gold nanoparticles, as a class of nanomaterials
with many unique properties such as colorimetric, conductivity, and nonlinear optical properties, have been found
many successful applications in biomolecular detection.[1921]
AuNPs have the advantages such as rapid and simple chemical synthesis, easy preparation in a wide range of sizes, for
example, approximately 1 to more than 100 nm. Furthermore, as an excellent biological marker, AuNPs can easily
conjugate with biomolecules and retain the biochemical activity of the labeled biomolecules, such as proteins and
DNA.[2225] So far, some methods have been developed by
integrating the advantageous properties of MBs and AuNPs
in immunoassays.[2628]

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FULL PAPER
As an important tumor marker, the concentration of
alpha-fetoprotein (AFP) in serum is related to the state of
many diseases, such as hepatocellular carcinoma, hepatoblastoma and other liver diseases.[2931] Thus, the determination of the AFP level is very helpful for clinical tumor diagnoses. So far, a variety of methods and strategies have been
proposed for the determination of AFP, such as radioimmunoassay (RIA),[32] enzyme-linked immunosorbent assay
(ELISA),[33] electrochemical sensors,[34] and fluoroimmunoassay (FIA).[35] However, only a few studies have been done
by using a chemiluminescence immunoassay (CLIA).[36] Due
to its high sensitivity and selectivity for immunoassay, CLIA
has been widely used in food,[37] environmental[38] and clinical assays.[39]
Among the CLIAs, the development of a highly sensitive
CLIA detection system for horseradish peroxidase (HRP) is
of great significance as it is important for the determination
of HRP-labeled antibodies in nonisotopic immunoassays.[40]
HRP is the enzyme that has gained the greatest significance
in analytical biochemistry for the determination of hydrogen
peroxide and other compounds through coupled enzymatic
reactions.[41, 42] A sensitive detection system of enhanced
CLIA from HRP-catalyzed oxidation of luminol has
become increasingly popular.[43] So far, a variety phenol derivatives have been applied as luminol signal enhancers,
which can significantly increase the intensity of the emitted
light.[44] Certain chemical indicators, such as phenolphthalein, cresolphthalein, phenol red, and so on, have also been
developed as enhancers of the HRP-catalyzed chemiluminescent oxidation of luminol.[45]
In this work, we present a sensitive and promising analytical method employing a new CL enhancer, bromophenol
blue (BPB), based on a an MB separation and AuNP labeling technique. This novel strategy takes advantage of easy
magnetic separation by MBs and amplification feature of
colloidal gold label along with CL intensity enhanced by
BPB for the luminolH2O2HRP system. In the proposed
new enhanced luminolH2O2HRPBPB CL system, the optimized CL reaction conditions and mechanism were discussed. As an alternative method of ELISA, the concentrations of AFP in human serum samples were detected by
sandwich-type immunoreaction under the optimum conditions.

Figure 1. Kinetic curves of luminol-H2O2-HRP chemiluminescence reactions a) enhanced by BPB and b) without enhancer. Conditions: luminol,
5.0  10 4 m in pH 11.0 NaOHNaHCO3 buffer solution; H2O2, 3.0 
10 4 m; HRP, 1.0  10 10 g mL 1; BPB, 8.0  10 5 m.

that without enhancer (Figure 1 b). These characteristics

showed the usefulness of BPB as a particle enhancer in luminolH2O2HRP CL system and, therefore, it could be applied to the sensitive determination of HRP and HRP-conjugates in immunoassay.
To determine the sensitivity of the luminolH2O2HRP
BPB system, the enhancement characteristics of BPB were
compared with that of the most conventional and widely
used CL enhancer, p-iodophenol (PIP), under the optimal
reaction conditions for each other (Figure 2, and see more
details from Supporting Information).[46, 47] The results
showed that BPB would be a more potent enhancer than
PIP with respect to the sensitivity for the determination of
HRP, and could facilitate a rapid quantification of HRP.

Results and Discussion

The kinetic characteristics of BPB enhanced luminol-H2O2HRP CL system: The kinetic curve of light emission obtained with BPB was compared with that without using enhancer in the luminolH2O2HRP system. As shown in
Figure 1, the CL emission with BPB reached a maximum
immediately after injection of luminol and decreased quickly under these analytical conditions. So a peak-shaped signal
was obtained and the CL intensity was defined as the peak
height of the CL kinetic curve. Furthermore, this CL intensity was largely enhanced by BPB (Figure 1 a) compared with

Chem. Eur. J. 2009, 15, 4704 4709

Figure 2. Kinetics of CL emission obtained with each enhancer. The concentration of HRP was 2.0  10 7 g mL 1. Top: Optimal CL reaction conditions for luminolH2O2HRPBPB system: 0.08 mm BPB, 0.3 mm
H2O2, 0.5 mm luminol in pH 11.0 NaOHNaHCO3 buffer solution.
Bottom: luminolH2O2HRPPIP system: 0.75 mm PIP, 3 mm H2O2,
0.15 mm luminol, all of these regents were prepared in pH 7.5, 0.1 m Tris
HCl buffer.

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S. Zhang et al.

Parameters affecting CLIA signals: For the sandwich immunoassay, certain parameters such
as the incubation times for immunoreactions would be expected to affect the sensitivity
of the method. Thus, after optimization of the luminolH2O2
HRPBPB CL reaction (See
details from Supporting Information), the effects of incubation times for the two immunoreactions (procedures III to IV
and IV to V in Scheme 1) were
examined with a solution containing 1.0 ng mL 1 of AFP.
For the incubation time between the primary antibody and
AFP sample (procedure III to
IV in Scheme 1), five incubation Scheme 1. The schematic illustration of the determination of AFP based on the sandwich-type chemiluminestimes of 5, 10, 20, 30 and 40 min cence immunoassay.
were studied. It was found that
the CL signal was almost constant by using a longer incubation time than 30 min (see Figure 3 a). Hence, 30 min of incubation time for the first immunoreaction was selected for the following experiments.
The incubation time between AFP sample and the secondary HRP-labeled antibody (procedure IV to V in
Scheme 1) was investigated from 0.5 to 3 h. As seen in Figure 3 b, with increasing incubation time, the CL signal increased and leveled off at 2 h. Consequently, subsequent
work employed 2 h as the incubation time for AFP sample
and the secondary antibody immunoreaction.
CLIA for the determination of AFP: According to the protocol shown in Va of Scheme 1, AFP was detected under the
optimized conditions. Figure 4 shows the calibration curve
obtained when the substrate solution (100 mL) containing
H2O2 and BPB, and luminol (200 mL) were injected into the
determination cuvette in which MBs (100 mL), incubated
with AFP sandwich immunostructure by employing AuNPs
as HRP-labels, had been placed. A peak-shaped signal was
observed within 30 s. A calibration curve in the AFP concentration range from 0.1 to 5.0 ng mL 1 showed a linear
correlation (R = 0.9997) represented by I = 2235.78C +
121.18, in which I is the peak height of CL and C is the concentration of AFP. The detection limit of 0.01 ng ml 1 of
AFP could be estimated by using 3s. A series of 11 measurements with AFP (0.2 ng mL 1) was used for estimating
the precision of the calibration curve and yielded a relative
standard deviation (RSD) was 8.2 %, indicating an acceptable level of precision.
At the same time, these results were compared with those
obtained by the method without using AuNPs as HRPlabels (Vb in Scheme 1). The linear range obtained from the
method was from 1.0 to 50.0 ng mL 1 with the detection
limit of 0.2 ng mL 1 (3s) (see Figure S3a Supporting Infor-

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Figure 3. Effects of incubation times on a) the first immunoreaction between anti-AFP monoclonal antibody on the MBs and AFP antigen and
b) the second immunoreaction between the primary antibody-AFP complex and the HRP-labeled secondary antibody. The concentration of
AFP antigen was 1.0 ng mL 1.

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Chemiluminescence Immunoassay

FULL PAPER
and it was confirmed that the excited-state 3-aminophthalate
anion was a luminophor.[45, 48] For the luminolH2O2HRP
BPB system, the enhanced CL spectra was obtained immediately after injection luminol into H2O2HRPBPB substrate solution (Figure 5).

Figure 4. The calibration curve for the determination of AFP using HRPlabeled anti-AFP antibody-modified AuNPs as labels. Inset was the amplification of the linear range from 0.1 to 1.0 ng mL 1 for AFP determination.

mation). In the case of the conventional ELISA for the determination of AFP by using an immunoplate, the linear
range was from 2.0 to 200.0 ng mL 1 with the detection limit
of 2.0 ng mL 1 (see Figure S3 b Supporting Information). So
the detection limit of this new enhanced CL system is one
order of magnitude lower than that without using AuNPs,
and much lower than that of ELISA.
Determination of AFP in human serum samples: To verify
the applicability of the present method to real samples, the
determination of AFP in human serum was conducted by
the present method and compared with those obtained by
the conventional ELISA method (Table 1). The comparative

Table 1. Determination of AFP in human serum samples.

Sample

Present
method [ng mL 1]

ELISA
ACHTUNGRE[ng mL 1]

Relative
deviation [%]

1
2
3
4
5
6
7
8
9
10
11

5.6
2.3
15.9
47.2
1.1
21.5
12.2
0.8
129.7
631.9
892.5

5.9
[a]
16.3
45.1

20.1
10.6

122.1
621.8
901.8

5.1

2.5
4.7

7.0
15.1

6.2
1.6
10.3

[a] The concentration of AFP could not be detected.

study between these two methods was also investigated, and

the results were shown in Figure S4 Supporting Information.
It can be seen that a relatively good correlation was obtained (R = 0.9999) between these two methods.
Mechanism for the enhanced CL system: The reaction
system of luminolH2O2HRP has been extensively studied,

Chem. Eur. J. 2009, 15, 4704 4709

Figure 5. CL spectra for luminolH2O2HRPBPB (c) and luminol

H2O2HRP (a) system.

It can be seen clearly that the emission wavelength was

around 425 nm, which was in good agreement with that of
luminolH2O2HRP system. Consequently, we could assume
that the luminophor for this new enhanced chemiluminescence system was also the excited-state 3-aminophthalate
anion, and the introduction of BPB did not result in generating of a new luminophor. Furthermore, to check whether
the superoxide ion (O2 C) and HOC played a role in the reaction, the effects of quencher superoxide dismutase (SOD)
and four HOC quenchers, tert-butanol, n-butanol, mannitol,
glycine, were investigated. From the results of Table 2, the
CL intensity was significantly inhibited by SOD and was
almost unchanged by introduction of all HOC quenchers.
Consequently, we assumed that O2 C was generated during
the reaction that did not involve HOC. The possible enhanced chemiluminescence mechanism is summarized in
Scheme 2.
Table 2. Effects of quenchers on luminolH2O2HRPBPB chemiluminescence system.
ICL

ICL
without quencher
100 mg mL 1 SOD
0.1 m tert-butanol

24 587
2408
23 932

0.1 m n-butanol
10 mm mannitol
10 mm glycine

24 343
24 889
24 574

Conclusion
A chemiluminescence immunoassay method for the sensitive and rapid determination of AFP is proposed by using
bromophenol blue as a novel enhancer through integration
with magnetic separation and colloidal gold-labeling tech-

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S. Zhang et al.

China).
Horseradish
peroxidase
(HRP) (  250 units mg 1 protein), Nhydroxysuccinimide (NHS), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) and bovine
serum albumin (BSA) were purchased
from Sigma. Superoxide Dismutase
(SOD) was obtained from Worthington Biochemical Corporation, and tertbutanol, n-butanol, mannitol, and glycine from Sinopharm Chemical Reagent (China). A luminol (standard
powder, Sigma-Aldrich) stock solution
(2.5  10 2 m) was prepared by dissolution in 0.1 m NaOH and further stored
in dark. The stock solution was consecutively diluted with 0.1 m NaOH/
NaHCO3 in order to obtain the proper
solution used for CL determination.
BPB was purchased from Shanghai
Aibi Chemistry Preparation (China).

Scheme 2. The mechanism of BPB enhanced luminolH2O2HRP chemiluminescence system.

niques. As a new chemiluminescence enhancer of luminol

H2O2HRP system, BPB has an advantage with respect to
the sensitivity in determining HRP after optimizing CL reaction conditions. Based on this new luminolH2O2HRP
BPB CL system, sandwich-type immunostructure MBs and
HRP-labeled anti-AFP antibody-modified AuNPs were employed for the determination of AFP, and a linear range of
AFP standard solution was from 0.1 to 5.0 ng mL 1 with the
detection limit of 0.01 ng mL 1 (3s). The application of this
method was also used to determine AFP in human serum
samples, and the results correspond well to those of conventional ELISA. This novel method demonstrated here may
open up new possibilities for biological assays and clinical
diagnoses.

Experimental Section
Apparatus and chemicals: The CL measurements were performed with a
BPCL ultraweak luminescence analyzer (Institute of Biophysics Academic Sinica, Beijing, China), and the CL spectra were measured on F-4500
fluorescence spectrophotometer (HITACHI, Japan). The ELISA results
were obtained by measuring the absorbance at 490 nm with the enzyme
microplate reader (DG3022 A , Nanjing, China). Transmission electron
microscopy (TEM) and scanning electron microscopy (SEM) images
were taken with JEM-2000EX/ASID2 and JSM-6700F instruments (HITACHI, Japan), respectively. UV/Vis spectra were carried out on a Cary
50 UV/Vis/NIR spectrophotometer (Varian). MBs modified with carboxyl groups (3.0  4.0 mm, 10 mg mL 1) and magnetic rack were obtained
from BaseLine Chrom Tech Research Centre (Tianjin, China).
The anti-AFP monoclonal antibody used as the capture antibody, HRPlabeled anti-AFP polyclonal antibody used as tracer antibody, and a
series of AFP standard solutions with various concentrations from 0.1 to
200.0 ng mL 1 were purchased from Biocell Bioeng. Co. (Zhengzhou,

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Doubly distilled water was used

throughout the experiments. All the
chemicals employed were of analytical
reagent grade and were used without
further purification. Human serum
samples provided by Qingdao Papermaking Hospital were stored at 4 8C.

Synthesis and characterization of Au

nanoparticles (AuNPs): AuNPs were
synthesized by following the method of reduction of tetrachloroauric acid
with trisodium citrate as reported by Ambrosi et al.[27] Briefly, an aqueous
solution of 0.01 % HAuCl4 (100 mL) was boiled with vigorous stirring,
and then an aqueous solution of 1 % trisodium citrate (3.5 mL) was
quickly added dropwise to the boiling solution. The color of the solution
turned from gray yellow to deep red, indicating the formation of AuNPs.
After cooling down the resulting colloidal suspension to room temperature with stirring, colloidal AuNPs were obtained. TEM were recorded
(see Figure S5 A Supporting Information) in order to confirm the average
size of AuNPs, and a UV/Vis spectrum was also recorded (see Figure S5 B Supporting Information).
Preparation of HRP-labeled antibody-conjugated AuNPs: The HRP-labeled AuNPs were prepared according to a modified literature procedure.[27] The conjugation process was carried out as follows: The HRP-labeled AFP antibody (10 % more than the minimum amount, a total of
8.8 mg mL 1 of antibody, which was determined by gold flocculation test;
see more details at Supporting Information) was added dropwise to a colloidal gold suspension (1 mL) at pH 9.0, followed by stirring at room
temperature for 30 min. Then, the conjugate was centrifuged at 4 500 g
for 3060 min, and the soft sediment was resuspended in 0.01 m PBS (containing 1 % BSA) solution and stored at 4 8C. The TEM of HRP-labeled
antibody conjugated AuNPs can be seen in Figure S6 from Supporting
Information.
Preparation of antibody-immobilized MBs (Ab-MBs): The anti-AFP antibody was immobilized on the MBs according to the specific protocol
provided by Zhang[12] and the schematic procedure can be seen from
Scheme 1 (steps I to III). The SEM of the MBs is shown in Figure S8 in
the Supporting Information. Briefly, a suspension of MBs (100 mL), in a
1.5 mL Eppendorf tube (EP tube), were separated from the solution on a
magnetic rack. The MBs were washed three times with 0.01 m imidazol
HCl buffer (pH 7.0; 3  200 mL) and then suspended to a final volume of
100 mL in the same buffer solution. A 0.2 m NHS solution (100 mL) and a
0.8 m EDC solution (100 mL) were added to the EP tube and the mixture
was incubated at room temperature for 30 min to activate the carboxylate
groups on the MBs. The MBs were then washed three times with the
above buffer solution (3  200 mL) and resuspended to a final volume of
100 mL. An aqueous solution of the anti-AFP antibody (1.0 mg mL 1;
100 mL) was then added and the resulting suspensions were allowed to

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Chemiluminescence Immunoassay

FULL PAPER

stand overnight at 25 8C for the immobilization of the anti-AFP antibody

on the surface of the activated MBs. Finally, the resulting Ab-MBs were
separated from the solution magnetically and resuspended in 1 % BSA
solution (100 mL) for 2 h to eliminate the risk of unspecific binding, and
then washed twice with the above buffer solution (2  100 mL).
The immobilization method was found to be sufficient for CL detection
(see more details in Supporting Information) and the amounts of antiAFP antibody immobilized on the MBs were estimated by a photometric
method (see Supporting Information).
Preparation of sandwich-type immunocomplex MBs: In the present
method, a sandwich immunoassay was adopted for the determination of
AFP. The scheme of the determination procedure is shown in Scheme 1.
A 100 mL aliquot of suspension containing anti-AFP antibody MBs was
washed with imidazolHCl buffer (200 mL), and resuspended in PBS
buffer (100 mL) containing target antigen at a series of various concentrations (0.1 to 200.0 ng mL 1) or human serum samples. After incubation
with delicate shaking at 25 8C for 30 min, the AFP antigen captured with
the Ab-MBs was magnetically separated to remove the supernatant and
then washed three times with the above buffer. HRP-labeled anti-AFP
antibody (100 mL) was added to the target-capturing anti-AFP antibody
MBs suspension and then incubated with mechanical shaking for 2 h at
25 8C. The precipitate was then separated on magnetic rack and washed
five times with imidazolHCl buffer (5  600 mL). Finally, the sandwichtype immunostructure MBs was resuspended in PBS (100 mL).
AFP detection assay by CLIA: After introduction of a suspension of
sandwich-type immunocomplex MBs (100 mL) into a quartz cuvette, a
substrate solution (100 mL) containing 3.0  10 4 m H2O2 and 8.0  10 5 m
BPB were pipetted into this cuvette. The CL reaction was triggered, 10 s
after the CL analyzer began to record, by injecting 5.0  10 4 m luminol
(200 mL) with a syringe through a septum. The kinetics of CL signals between 0 to 30 s were recorded and the peak heights of the emission
curves were measured by means of a photon counting unit. In terms of
method sensitivity, AFP was also determined without using AuNPs, and
compared to the results of classical ELISA.

Acknowledgements
This work was supported by the National Natural Science Foundation of
China (No.20775038), the National High-tech R&D Program (863 Program, No. 2007AA09Z113), and the Excellent Young Scientists Foundation of Shandong Province (JQ200805).

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 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: August 19, 2008

Revised: November 18, 2008
Published online: March 16, 2009

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