Chinese-German Journal of Clinical Oncology March 2008, Vol. 7, No.

3, P179P184
DOI 10.1007/s10330-007-0188-z

Advances in the application of quantum dots in tumor

markers investigation*
Chuang Chen1, Liangdong Chen1, Zhiling Zhang2, Yan Li1
1
Department of Oncology, Zhongnan Hospital & Cancer Center, Wuhan University, Wuhan 430071, China
2
College of Chemistry & Molecular Sciences, Wuhan University, Wuhan 430072, China

Received: 30 November 2007 / Revised: 25 December 2007 / Accepted: 12 January 2008

Abstract Tumor markers have been of vital importance in cancer diagnosis, treatment and monitoring. However, the sen-
sitivity of current tumor markers for early diagnosis is low, reducing the clinical usefulness of tumor markers. Quantum dots
are new fluorescent nanoparticles with unique photophysical and chemical properties, thus having a great potential impact on
the investigation of cancer pathogenesis, early diagnosis, targeted therapy, prognosis and monitoring, when combined with
tumor markers. The current research is focused on the detection of specific tumor markers or molecules based on tangible
carriers such as cells and tissues. One of the most promising clinical applications would be to explore the potential of this
highly sensitive labeling technique for the detecting and imagining tumor markers in serum and other body fluids, where some
progresses have already been made recently. How to detect early cancer based solely on invisible carriers would be the next
step of quantum dots bio-probes in clinical use, so as to develop a new detection technique with greater sensitivity, specificity,
rapidity and availability.

Key words quantum dots; tumor markers; bio-probes; biomedical application; cancer

Cancer has become one of the most serious global
health threats. According to the China Healthcare Statis-
tics Report, 2006 [1], cancer has become the number one
cause death in 2006, accounting for more than 25% of all
deaths; and cancer has also become the first disease killer
for Americans under the age of 85 years old since 1999
[2]
. In the anti-cancer campaign, tumor markers (TMs)
have played an important role in the diagnosis, progno-
sis, treatment selection and monitoring of cancer, and a
series of TMs for clinical use have been approved by FDA
recently [3]. However, the sensitivity of TMs for early di-
agnosis needs to be greatly improved. For example, the
12-tumor-marker protein chip system (C12) developed in
China [4] is a newly diagnosis system for a wide range of
malignant tumors and has been extensively used in clini-
cal practice in recent years. The evaluation of this system
for the diagnostic value in gastric and colorectal can-
cer has been conducted recently [5, 6] and the key results
were summarized in Table 1. The overall diagnostic rate
Correspondence to: Yan Li. Email: liyansd2@163.com
* Supported by the grants from the New-Century Excellent Talents Sup-
porting Program of the Ministry of Education of China (No. NCET-04-
0669), the Foundation for the Author of National Excellent Doctoral Dis-
sertation of China (No. 200464), the Wuhan Innovation Study Project (No.
20066002054), the Natural Science Foundation of China (No. 20675058)
and the Science Fund for Creative Research Groups (No. 20621502),
NSFC.
of C12 biochip system in our group of 130 patients with
colorectal cancer was 42.31%, and only 13.64% for stage
I. And the result was even worse in the gastric cancer
group, with overall diagnostic rate 37% and only 7.8%
for stage I. In addition, how to incorporate the increas-
ing information of TMs into the determination of cancer
diagnosis, staging, prognosis and monitoring was another
important issue [3], which was one of the reasons why
many TMs have been found but were not widely used in
clinical practice [7]. Therefore, the promising clinical uses
of TMs should be concentrated on exploring a new detec-
tion technique with greater simplicity and sensitivity and
establishing a more reasonable application platform for
monitoring. Fortunately, with the development of nano-
technology [8], especially the new fluorescent nanopar-
ticles, quantum dots (QDs), as probes for biomedical ap-
plication will bring new promising to cancer research [9],
and the combination with TMs has been one of the hot
issues in recent years [10]. Advances for this technique in
TMs investigation were reviewed in this article.
Properties of quantum dots
QDs are new semiconductor nanocrystals with sizes
ranging from 1 nm to 10 nm in diameter. The special
physical composition and size lead to many unique bio-
medical properties, such as higher fluorescence intensity,
180 www. springerlink. com/content/1613-9089

Table 1 Values of TM biochip C12 system in the diagnosis of gastric
and colorectal cancer*
Gastric cancer (%) Colorectal cancer (%)
Items (n = 100) (n = 130)
Overall diagnostic rate 37.0 42.1
Diagnostic of stage I 7.8 13.6
Diagnostic of stage II 29.4 39.5
Diagnostic of stage III 35.5 38.2
Diagnostic of stage IV 50.0 68.8
* One or more tumor makers beyond the normal range were considered
to be positive
longer fluorescence lifetime, sensitive detection of QDs
signals over intrinsic biological fluorescence and simul-
taneous detection of many biomarkers [11]. Furthermore,
appropriate composition and size of QDs can emit near
infrared optical spectrum (7002000 nm) which has low
tissue scatter and absorption, so as able to obtain optical
signals of maximized penetration depth from biological
tissue, and was ideal to deep-tissue imaging, especially in
vivo imaging [12]. The rapid development of QDs in bio-
medical applications was intimately associate with the in-
creasing progresses on the synthesis and bio-conjugation
of QDs in recent years [1317], especially the application
of conjugation by PEG (polyethylene glycol), which not
only makes QDs water soluble and stable, but also make
them escape recognition and non-specific uptake by re-
ticuloendothelial system (RES), and thereby prolonging
the half life of QDs in circulation [18, 19].
The application of QDs probes in
TMs investigation
Since Bruchez et al [20] and Chan et al [21] demonstrated
that QDs can be used as a bio-probe to label live cells,
many important encouraging developments in biomedical
field [15, 22, 23] have been made, in which the combination of
QDs probes with TMs has demonstrated many promising
advantages [911]. Table 2 summarizes the current applica-
tion of QDs in some TMs investigations [3] as approved by
US Food and Drug Administration (FDA).
HER2
HER2 (human epidermal growth factor receptor 2) is
one of the most widely used TMs in clinical practice and an
important prognosis factor of breast cancer after estrogen
and progesterone receptors [24, 25], as well as an important
indicator to guide the use of Trastuzumab (Herceptin), a
monoclonal antibody in molecular targeted therapy [26].
Therefore, this TM has become one of the important TMs
in the application of QDs.
Cellular recognition
Wu and coworkers [27] demonstrated that HER2 over-
expressing human breast cancer cell line SK-BR-3 had
been successfully and specifically recognized by two
kinds of QD-IgG probes after incubating with a mono-
clonal HER2 antibody; then they used the QD-streptavi-
din probe instead of QD-IgG probe, which was not only
specific enough to recognize the antigen of HER2 but the
fluorescence intensity was even greater. More significant-
ly, they successfully achieved imaging HER2 and nuclear
antigen simultaneously in the same optical excitation by
using two different QD-IgG streptavidin probes, together
with a monoclonal HER2 antibody, antinuclear antibody
and their homologous IgG. This study not only proved
that QD probes had enough specificity and fluorescence
intensity, able to recognize the molecule target at sub-
cellular level, but also got multi-fluorescent imaging of
subcellular structure, thus possible to observe the TM in
static or dynamic state simultaneous in the same opti-
cal excitation. The HER2 over-expressing human breast

Table 2 Application of QDs in TMs investigations as approved by FDA

Guidance of FDA Application of QDs
Types of TMs
Source Cancer type Clinical use Source Cancer type Application
HER2/neu* Tumor tissue and Breast cancer Prognosis, selection Serum, cell and Breast cancer Cellular recognition
serum of therapy and tumor tissue and molecular and
monitoring in vivo imaging
PSA** Serum Prostate cancer Screening and Serum, cell and Prostate cancer Cellular recognition,
monitoring tumor tissue in vivo imaging and
serum diagnosis
Epidermal growth Tumor tissue Colon cancer Selection of therapy Cell and tumor Uterine cervix Cellular recognition
factor receptor tissue cancer and signal
transduction
)HWRSURWHLQ Serum Nonseminomatous Staging Cell and tumor Liver cancer Cellular recognition
testicular cancer tissue and in vivo imaging
CA125 Serum Ovarian cancer Monitoring Cell and tumor Ovarian cancer Cellular and tissular
tissue recognition
* In the guidance of FDA, source of serum was used for monitoring and tumor tissue used in prognosis and selection of therapy; ** Guidance of FDA
included total and complex PSA, and the application included PSMA which was a new PSA and was used in cellular recognition and in vivo imaging
Chinese-German J Clin Oncol, March 2008, Vol. 7, No. 3
cancer cell line KPL-4 is sensitive to trastuzumab which
is a chimeric monoclonal antibody against HER2. In one
study, three different sizes of QDs labeled with trastu-
zumab can successfully recognize KPL-4 cells but not
recognize the HER2 low-expressing human breast cancer
cell line MDA-MB-231. The fluorescence intensity of the
KPL-4 cell line was ten times as high as MDA-MB-231
cell line, corresponding to the different level of HER2 ex-
pression on the surface of the two cell lines. This study is
expected to have good application in molecular targeted
therapy in breast cancer. Yu et al [19] used the PEG modi-
fied QDs probes and successfully targeted to the SK-BR-3
breast cancer cells with HER2 receptor. This study fur-
ther demonstrated that the PEG modified QDs probes can
limit nonspecific binding and increase the binding effi-
ciency, and also documented that the specific recognition
was indeed from the antibody-antigen interaction.
Non-viral vector study
Chitosan nanoparticle can be used as a new non-vi-
ral vector for gene therapy [29]. In one study [30], chitosan
nanoparticles encapsulating QDs were synthesized and
conjugated with monoclonal HER2 antibody. Using this
construct, HER2/neu siRNA can be transfected into HER2
over-expressing SKBR3 breast cancer cells by recognizing
the HER2 receptor, and the gene-silencing effects was also
identified using the luciferase and HER2 ELISA assays.
This study demonstrated that chitosan nanoparticles with
encapsulated QDs had higher transfection efficiency, and
also more accurate targeted recognition, which was one
of the directions of gene therapy of breast cancer.
Mechanism of drug transport
Drug treatment was one of the important components
of breast cancer treatment. Tada et al [31] reported that
QDs conjugated with monoclonal HER2 antibody were
injected into living mice model of breast cancer with
HER2-overexpressed and analyzed the processes of its
mechanistic delivery to the tumor. They successfully ob-
served the six processes of delivery: initially in the circu-
lation within a blood vessel, during extravasation, in the
extracelullar region, binding to HER2 on the cell mem-
brane, moving from the cell membrane to the perinuclear
region, and in the perinuclear region. This study will be
helpful for studying the mechanism of drug transport and
enhancing therapeutic efficacy.
PSA
PSA level is associated with the presence of prostate
cancer, and can be used as a screening marker of prostate
cancer [32]. Taneja et al [33] found that PSA was associated
with the staging of prostate cancer, while PSMA (pros-
tate-specific membrane antigen) was a new maker of PSA
and can be used as an important supplement of PSA.
181
In vivo imaging
Gao et al [13] synthesized a specially modified mul-
tifunctional QDs probe to realize simultaneous cancer
targeting in a mice xenograft model of prostate cancer.
This modified core-shell CdSe/ZnS QDs probe contained
an ABC triblock copolymer which was able to prevent
particle aggregation and fluorescence loss, monoclo-
nal antibodies for PSMA recognizing and multiple PEG
molecules for enhancing biological efficacy. First, using
this probe they successfully recognized the PSMA over-
expressing of human prostate cancer cell line C4-2, then
the probes were injected into a normal mouse and a tu-
mor-bearing mouse respectively through the tail vein.
They had achieved fluorescence imagines in tumor bear-
ing mice and found that the QDs probes targeted and
accumulated at tumor sites and the fluorescence signals
were obtained, with both intensity and duration higher
than that of green fluorescence protein (GFP); while in
the control group, there were no significant fluorescence
signals, completely covered up by auto-fluorescence and
background. Further studies indicated that this active tar-
geting by tumor-specific QDs were much faster and more
efficient than passive targeting based on tumor perme-
ation, uptake and retention. More recent study by this
group [14] indicated that multicolor fluorescence imaging
in vivo prostate mouse model can be achieved by only us-
ing as few as 10100 cancer cells, which was the base of
detection of genes, proteins, and small-molecule drugs in
single living cells with QDs probes, and this spectral im-
aging modality can be adopted for real-time visualization
of cancer cell metastasis in live animals.
Serum diagnosis
One of the most promising clinical applications would
be to explore QDs probes for the detection of tumor
markers in serum [10]. Azzazy et al [34] using QDs strep-
tavidin conjugates detected biotinylated PSA at 0.00038
ng/mL. Recently, Kerman et al [35] reported that total PSA
(TPSA) in human serum can be detected successfully by
QDs streptavidin conjugates and biotinylated monoclonal
antibody complex using a sandwich assay approach, and
the detection limit was up to 0.25 ng/mL, with a wide
range of detection from 0.25 ng/mL to 100 ng/mL.
EGFR
EGFR (epidermal growth factor receptor) is over-ex-
pressed on surface of many malignant tumor cells, such
as colon cancer, breast cancer, uterine cervix cancer and
lung cancer [36], and the level of EGFR can be increased at
an early stage of tumorigenesis.
Cellular recognition
Nida et al [37] observed that the anti-EGFR QDs con-
jugates can specifically label EGFR over-expressing SiHa
182
cervical cancer cells. The imaging was performed on a
laser scanning confocal microscope with two kinds of ex-
citation and the fluorescence intensity was both higher as
compared with control group. On the basis on this study,
Rahman et al [38] achieved an imaging of the three-dimen-
sional tissue constructs of SiHa cervical cancer cells by
using the same methods, and the results were also similar.
Furthermore, this study suggested that the novel optical
contrast agents was superior to traditional contrast agents
in respect of the identification of tumor cells from intact
tissue; and also further documented that the QDs probes
can be used in detect cervical cancer on molecular level,
providing a new approach in the early detection of many
cancers.
Study of signal transduction mechanism
Thorough understanding of signal transduction mech-
anism is the basis of targeted therapy. Lidke et al [39] had
successfully achieved the detection of erbB/HER recep-
tor-mediated signal transduction process using QDs tech-
niques. First, they documented that the affinity of QDs to
EGF remained stable after combination and was able to
actively bind to erbB1 receptor by endocytosis. The pro-
cess of combination and signal transduction between EGF
and erbB1 could be traced with QDs probes. The entire
dynamic cellular processes were monitored in real-time,
which included the signaling molecule binding to the
membrane, bound to cell filopodia, endocytosing inter-
nalization and the interaction with erbB2 and erbB3. This
study documented that cell filopodia indeed had a newly
mechanism of retrograde transport to the cell body, while
this process could previously only be studied on fixed
cells or by biochemical fractionation.
AFP
Chen et al [40, 41] and Yu et al [42] had achieved immu-
no-fluorescence imaging of AFP over-expressing human
hepatocellular carcinoma cell lines HCCLM6, using AFP
monoclonal antibody and QDs-IgG probes, which was the
conjugation of thioglycolic acid modified water soluble
core-shell CdSe/ZnS QDs and the corresponding second-
ary antibody (Fig. 1). In vivo tumor xenografts and lung
metastases models were then established by inoculation
of HCCLM6 cells subcutaneously and into the tail vein
of nude mice, respectively. The live animal fluorescence
imaging was achieved successfully by using the QD-AFP-
Ab probes (Fig. 2). Further analysis on the probes distri-
bution in tumor showed that the fluorescence of QDs
was not homogeneous in tumor, and the probes per field
were lower in the center than in the periphery of the tu-
mor, indicating that the proliferation in different tumor
sites was not similar (Fig. 3). Non-specific uptake of QDs
probes occurred mainly in the liver, spleen and lungs.
www. springerlink. com/content/1613-9089
CA125
CA125 is a useful TM in the monitoring and treatment
of ovarian cancer [43], and is one of the FDA approved TMs
that can be used in clinical in vivo immuno-imaging for
the detection of tumor metastases [3]. In one report [44], the
level of CA125 was detected in human ovarian cancer cell
lines HO8910, human ovarian carcinoma tissue and tumor
xenografts in mice by using streptavidin QDs together
with biotinylated monoclonal antibody. This probe had
more sensitivity, higher fluorescence intensity and longer
duration compared to the FITC labeled probes.
Potential problems
Several problems should be solved before clinical ap-
plication of QDs probes. First is the toxicity issue of QDs
[45]
. The cadmium and selenium in QDs are toxic to many
culture cells. Although Dubertret et al [46] documented
that appropriately modified QDs had no discernible effect
on cellular physiological activities in short time, includ-
ing cell growth, cell division, signal transduction and cell
movement, Chang et al found [47] that the cytotoxicity of
QDs was associated with the intracellular level of QDs by
cellular endocytosis rather than the extracellular level. In
addition, the shell of QDs can be damaged by oxidation
and ultraviolet irradiation, thereby releasing toxic heavy
metals. Therefore, the long-term toxicity of QDs should
be further investigated.
Other problems
Although the fluorescent properties of QDs are superi-
or to traditional fluorescent dyes, several other problems
except for toxicity should also be addressed [9, 15, 48, 49]: (1)
The size of QDs is larger than traditional fluorescent dyes,
and even larger after modification with other functional
groups, thereby leading to a significant steric hindrance,
which limits its biological applications; (2) The different
approaches to surface modification and crosslinking of
QDs have effects on the physical and chemical properties
of QDs, decreasing the quantum yield; (3) The blinking
was another annoying characteristic of QDs, and the sta-
bility of single QDs are still to be improved. In addition,
the quantitative relations between soluble modified QDs
and functional groups (e.g. antibody and biotin) are still
to be determined.
Summary and promising
With the advancements in genomics and proteomics,
greater numbers of more specific TMs will be discovered
and applied to clinical use. However, the current tradi-
tional fluorescent dyes such as FITC (fluorescein isothio-
cyanate) and rhodamine have limited use in the study
of interaction of dynamic biomolecules in real-time and
Chinese-German J Clin Oncol, March 2008, Vol. 7, No. 3
Fig. 1 The confocal microscopic images of HCCLM6 cells with QD-IgG
probes. AFP in the cytoplasm was recognized by QD-IgG probes. There
was an obvious binding of QD-AFP-Ab probes to the tumor cells. Nuclei
ZHUHFRXQWHUVWDLQHGEOXHZLWK'$3,6FDOHEDU P$GDSWHGIURP
Chen et al [40, 41] with permission from the publisher)
Fig. 2 In vivo targeting and imaging tumor with QD-AFP-Ab probes. (a)
The whole body and ex vivo imaging of tumor bearing nude mice. The QD
fluorescence was localized in the tumor site; (b) The spectra analysis of
the tumor site (red arrow) and the normal tissue adjacent to the tumor site
(green arrow). The fluorescence spectra of tumor site were the same with
those of QD-AFP-Ab probes, but there was no characteristic 590 nm peak
of the QD at the normal tissue site; (c) The confocal microscopic imaging
of the tumor section showed the specific binding of QD-AFP-Ab probes to
WXPRUFHOOV6FDOHEDU P$GDSWHGIURP&KHQet al [41] with permis-
sion from the publisher)
the diagnosis of tumor at early stage [50]. QDs with unique
photophysical and chemical properties have a great po-
tential impact on cancer pathogenesis, early diagnosis,
targeted therapy, prognosis and monitoring by combin-
ing with TMs. The current research is focused on the de-
tection of specific tumor markers or molecules based on
tangible carriers such as cells and tissues. One of the most
promising clinical applications would be to explore the
potential of this highly sensitive labeling technique for
the detecting and imagining of TMs in serum and other
183
Fig. 3 QD-AFP-Ab Probes Distribution in Tumor. The site-by-site spec-
tra analysis of tumor showed that the probes per field were lower in the
center than in the periphery of the tumor, indicating that the growth of
tumor was not homogeneous and the peripheral site was more active.
(Adapted from Chen et al [41] with permission from the publisher)
body fluids. For example, using this technology, Azzazy et
al [34] and Kerman et al [35] had detected the level of PSA in
serum and the detection limit were up to 0.00038 ng/mL
and 0.25 ng/mL, respectively. How to detect early cancer
based solely on invisible carriers would be the next step
of quantum dots bio-probes in clinical use, so as to de-
velop a new detection technique with greater sensitivity,
specificity, rapidity and convenience.
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