Biomaterials 34 (2013) 4274e4283
Contents lists available at SciVerse ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Bimodal optical diagnostics of oral cancer based on Rose Bengal conjugated gold
nanorod platform
Jia-Hong Wang a,1, Beike Wang b,1, Qian Liu b, Qian Li a, Hao Huang a, Li Song b, Tian-Ying Sun a,
Huaiyu Wang c, Xue-Feng Yu a, c, *, Chengzhang Li b, **, Paul K. Chu c, ***
a
Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan 430072, China
The State Key Laboratory Breeding Base of Basic Science of Stomatology, Hubei-MOST & Key Laboratory of Oral Biomedicine of Ministry of Education,
Department of Periodontics, School and Hospital of Stomatology, Wuhan University, Wuhan 430079, China
c
Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 January 2013
Accepted 6 February 2013
Available online 13 March 2013
Early detection of cancer often requires time consuming protocols and expensive instrumentation. To
address these limitations, a Rose Bengal conjugated gold nanorod (RB-GNR) platform is developed for
optical detection of cancer cells. The GNRs are modified by poly(allylamine hydrochloride) and conjugated with RB molecules to produce RB-GNRs which exhibit strong optical absorption in the nearinfrared (NIR) region, good stability in aqueous solution, low cytotoxicity, and high specificity to oral
cancer cells. The label-free sensing assay utilizes RB-GNRs as the sensing probe and by monitoring the
aggregation-induced red-shift in the NIR absorption wavelength, specific and quantitative analysis of the
oral cancer cell lysate is accomplished down to a detection limit of 2000 cells/mL. By employing the RBGNRs as an imaging probe, an imaging assay is established on a home-made NIR absorption imaging
system. Based on the NIR absorption by the RB-GNRs specifically conjugated with the oral cancer cells,
multi-channel, rapid and quantitative detection of oral cancer cells is demonstrated. The high sensitivity
and specificity of the RB-GNR platform as demonstrated by the two complementary assays provide noninvasive optical diagnostics of oral cancer cells enabling convenient screening and monitoring.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Gold nanorods
Rose Bengal
Oral cancer
Optical diagnostics
Biosensors
Absorption imaging
1. Introduction
As a typical kind of epithelial cancer, oral cancer is the sixth
most common cancer in both men and women worldwide and the
5-year survival rate of patients suffering from the disease is only
about 50% [1,2]. The common symptoms during the early stage of
oral cancer include asymptomatic, innocuous-appearing mucosal
alteration which can also be a manifestation of a variety of mucosal
diseases [3]. Hence, although it may be precancerous, it is difficult
to distinguish and there is an urgent need for early detection of
cancer in order to improve the survival rate and achieve minimal
dysfunctions. Unfortunately, present screening tools are
* Corresponding author. Key Laboratory of Artificial Micro- and Nano-Structures
of Ministry of Education, School of Physics and Technology, Wuhan University,
Wuhan 430072, China. Tel.: þ86 27 68752481; fax: þ86 27 68752569.
** Corresponding author. Tel.: þ86 27 87646697; fax: þ86 27 87873260.
*** Corresponding author. Tel.: þ852 34427724; fax: þ852 34420542.
E-mail addresses: yxf@whu.edu.cn (X.-F. Yu), l56cz@hotmail.com (C. Li),
paul.chu@cityu.edu.hk (P.K. Chu).
1
J.-H. Wang and B. Wang contributed equally to this work.
0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biomaterials.2013.02.012
insufficient [4] and no effective monitoring methodologies are
available. A technique which is cost-effective and can be implemented on simple instrumentation while delivering the necessary
specificity and sensitivity is of scientific and clinical interest. In this
respect, optical techniques which can offer non-invasive and realtime diagnostics of diseased cells and tissues have attracted much
attention [5e16]. Compared to common diagnostic methods such
as CT, MR, and PET, optical techniques tend to be more convenient,
sensitive, specific, and independent of expensive instruments
[11,12,15]. In particular, near-infrared (NIR) light (650e950 nm) can
penetrate deeply into cells and tissues because of small absorption
by hemoglobin, fat, water, and so on thus creating the so called “NIR
biological window” [17]. Therefore, synthesis of the proper NIRoptical probes and development of the corresponding NIR-optical
diagnostic systems can increase the sensing and imaging sensitivity in cancer detection [6,12,18e23].
Gold nanorods (GNRs) have received much attention in medical
diagnostics due to their unique surface plasmon resonance (SPR)
properties in the NIR region [24e32]. As a typical one-dimensional
(1D) nanostructure, the GNRs exhibit strong distance-dependent
J.-H. Wang et al. / Biomaterials 34 (2013) 4274e4283
SPR properties as a result of plasmonic coupling [33]. Slight variations in the environment around the GNRs can induce significant
changes in the longitudinal SPR (LSPR) peak wavelength, which is
much more sensitive than the transverse SPR (TSPR) peak wavelength observed from spherical gold nanoparticles [34]. Therefore,
GNRs are potentially efficient probes to sense specific target binding events on biological samples [10,35e37]. Moreover, GNRs have
very strong optical extinction in the NIR LSPR band. The extinction
coefficient of GNRs is about 6 109 M1 cm1 that is much larger
than the value of 2.7 108 M1 cm1 of gold nanoparticles and
several orders of magnitude bigger than those of traditional organic
chromospheres [38]. Therefore, GNRs can also be used as direct NIR
absorption imaging probes in optical diagnostics [39].
Nanoparticles usually target cells by receptor-mediated binding
or endocytosis and a nanoparticle surface modified with a ligand
can efficiently bind with target cells. Surface functionalization of
gold nanoparticles by conjugation with a specific agent such as
aptamer, peptide, antibody, and protein has been proposed for
diagnosis and thermo-phototherapy of cancer cells [40]. With regard to oral cancer, Rose Bengal (RB), the 4,5,6,7-tetrachloro20,40,50,70-tetraiododerivative of fluorescein, is a specific agent of
cancer cells [41e45]. RB has been shown to have specificity to oral
cancer through inhibition of DNA polymerases of oral cancer cells
[41]. Based on its specificity, the RB molecule has been used as a
photosensitizer in fluorescence detection of oral cancer [43]. RB has
also been employed as a staining molecule to detect oral precancerous and malignant lesions clinically with colorimetric evaluation and high sensitivity and specificity of 93.9 and 73.7%,
respectively, have been demonstrated [44]. Furthermore, the RB
molecule is a suitable agent for nanoparticle bio-conjugation,
typical photosensitizer for photodynamic therapy (PDT) [46e51],
and probe for surface-enhanced Raman scattering (SERS) [52]. In
this paper, we describe the method to conjugate GNRs with RB
molecules and demonstrate that this RB-GNR platform is an
excellent NIR-optical diagnostic tool for oral cancer.
2. Experimental section
2.1. Synthesis and characterization of RB-GNR probes
2.1.1. Materials
Chloroauric acid (HAuCl4$4H2O, 99.99%), silver nitrate (AgNO3, 99.8%), L-ascorbic
acid (99.7%), sodium chloride (NaCl, 96.0%), and hydrochloric acid (HCl, 36e38%)
were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Sodium borohydride (NaBH4, 96%), poly (allylamine hydrochloride) (PAH,
MW w 15,000 g/mol), and RB (95%) were obtained from Aldrich (America). Hexadecyltrimethylammonium bromide (CTAB, 99.0%) was purchased from Amresco
Inc. (America). All the chemicals were used as received without purification. Ultrapure water with a resistivity of about 18.25 MU cm was used as the solvent in all
the experiments.
2.1.2. Synthesis of GNRs
The GNRs were synthesized in an aqueous solution using a seed-mediated
growth method [24,28]. The 3e4 nm gold seed particles were prepared by mixing
5 mL of 0.5 mM HAuCl4 with 5 mL of 0.2 M CTAB. The solution was stirred vigorously
followed by drop wise addition of 600 mL freshly prepared ice-cold 10 mM of NaBH4.
The seed solution was left for more than 2 h before use. In the GNR synthesis, 18 mL
of 5 mM HAuCl4 and 225 mL of 0.1 M AgNO3 were added to 90 mL of 0.2 M CTAB and
then 200 mL of 1.2 M HCl and 10.5 mL of 10 mM ascorbic acid were added and gently
swirled as the color changed from dark orange to colorless. After the color had
changed, 120 mL of the CTAB-stabilized gold seed solution was rapidly injected. The
resulting solution was gently mixed for 10 s and left undisturbed overnight. Finally,
the GNR solution was centrifuged at 12,000 rpm for 15 min to stop the reaction. The
supernatant was removed and precipitate was resuspended in ultrapure water. The
GNR concentration was estimated to be about 0.65 nM according to extinction coefficient at the LSPR wavelength [53].
2.1.3. Synthesis of RB-GNR probes
The GNRs were coated with PAH using a method previously described by
Murphy et al. [54]. 20 mL of 10 mg/mL PAH in 1 mM NaCl and 10 mL of 10 mM NaCl
were added to 100 mL of 0.65 nM GNRs and stirred for several hours at room
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temperature. Centrifugation cycle of 10,000 rpm for 10 min was performed to
remove the excess PAH and NaCl, obtaining PAH-coated GNRs. Then, 100 mL of the
PAH-coated GNRs aqueous solution was mixed with 100 mL of 10 mM RB aqueous
solution and the mixture was kept in 30 C for at least 3 h. Afterwards, the samples
underwent centrifugation at 10,000 rpm for 10 min to remove the excess RB, and redispersed in 100 mL of ultrapure water. The concentration of the RB-GNRs was about
0.5 nM.
2.1.4. Characterization
Transmission electron microscopy (TEM) images were taken on a JEOL 2010
transmission electron microscope at an accelerating voltage of 200 kV. The zeta
potential and hydrodynamic distribution of the samples was determined using a
Zeta sizer (Nano ZS90, Malvern Instruments, UK) at 25 C. The samples were
dispersed in deionized water with concentration of 0.5 nM. Absorption spectra were
taken on a TU-1810 UV-Vis-NIR spectrophotometer (Purkinje General Instrument
Co. Ltd. Beijing, China). Photoluminescence emission spectra were recorded on a
Hitachi F-4500 fluorescence spectrophotometer with a Xe lamp as the excitation
source.
2.2. Cell cultures
Two oral squamous cell carcinoma (OSCC) cell lines were used in this study. The
human OSCC cell line, CAL-27 (CRL-2095, ATCC), was supplied by the Affiliated Ninth
People’s Hospital, Shanghai Jiaotong University, Shanghai Research Institute of
Stomatology, Shanghai, China. The human OSCC cell line, Tca8113 (SCC from human
tongue), was purchased from China Center for Type Culture Collection (CCTCC in
Wuhan University). Both cell lines were cultured in Dulbecco’s modified eagle
medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco,
Carlsbad, California, USA) in a humidified atmosphere of 95% air and 5% CO2 at 37 C.
The protocols for obtaining human oral mucosa samples were reviewed and
approved by the Ethics and Investigation Committee of the School & Hospital of
Stomatology, Wuhan University. Patients with cleft lip and palate who received
treatment at the Department of Oral and Maxillofacial Surgery, School and Hospital
of Stomatology, Wuhan University were given sufficient prior information about this
study, and written consent was obtained from patients (1 male and 2 female with a
mean age of 1.5 years). The tissues from oral mucosa were harvested during oral
surgery. The mucosal sample was put in a 15 mL conical tube containing 5 mL of
serum-free Dulbecco’s modified eagle medium containing 1% antibiotice
antimycotic (Invitrogen). The primary oral keratinocyte cultures were established
and cells were serially passaged as previously described with only minor modifications [13]. The collected oral mucosa tissues were digested with dispase (2.4 mg/
mL, Boehringer Mannheim, Indianapolis, Ind.) overnight at 4 C. After incubation,
the oral keratinocytes were mechanically dissociated from the underlying connective tissue and the surface epithelium was digested further in 0.125% EDTA trypsin
(GIBCO, Buffalo, N. Y.) for 10 min at 37 C. After centrifugation at 1000 rpm for 5 min,
the cell pellets were collected and resuspended in the keratinocyte growth medium
(KGM-2; Clonetics, San Diego, Calif.). The cultures were incubated at 37 C under 5%
CO2 until confluence and passages 3e5 were used experimentally.
2.3. Cytotoxicity evaluation
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
tetrazolium reduction assay was used to measure the cytotoxicity of the RB-GNR
probes on OSCC cells CAL-27. The cancer cells were distributed in 96-well plates
at 1 104 cells/well and allowed to attach overnight. In the treatment of the RB-GNR
probes, the culture medium was replaced with one containing various concentrations of the RB-GNR probes from 0 to 0.45 nM and incubated at 37 C. After 4 h, the
medium was removed and the cells were rinsed twice with PBS. The wells were
refilled with the complete medium and the cells were incubated for another 24 h.
Subsequently, the cells were incubated in a culture medium with the MTT solution
(5 mg/mL) at 37 C and 5% CO2 for 4 h. The cells were lysed and blue formazan
crystals were dissolved in 150 mL of DMSO. The optical densities (OD) were
measured at 490 nm on an enzyme-linked immune sorbent assay (ELISA) platereader. Three wells were used for each concentration. The percentage of surviving
cells was calculated using the following formula: (OD treated/OD control) 100 and
three independent experiments were conducted.
2.4. SPR sensing of cancer cell lysates
2.4.1. Preparation of cell lysates
The OSCC cell lines CAL-27, Tca8113, and primary human oral epithelial cells
were plated on 10 cm diameter tissue culture dishes until confluence. The confluent
cultures of all the cells were incubated with 0.25% trypsin for 2 min, carefully detached by a cell scraper, washed twice in PBS, and resuspended in deionized water at
a density of 1 107/mL. The cell lysate was frozen in liquid nitrogen and thawed at
37 C using four freezeethaw cycles. To remove the crude cell debris, the lysate was
centrifuged at 5000 rpm for 15 min. The supernatant of the cell lysate was sampled
independently by a 1.5 mL peptite and stored at 70 C until use. The corresponding
cell debris was also collected as the control sample in the sensing experiments.
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2.4.2. Sensing experiments
The cell lysates of CAL-27 and Tca8113 cancer cells, and primary human oral
epithelial cells were used as the sensing samples. Different volumes (10, 30, 50, 70,
100, and 150 mL) of the cell lysates with cell destiny of 1 106/mL were added to
4.5 mL of 0.5 nM RB-GNR solution. The corresponding cell concentrations were
2.2 103, 6.5 103, 10.7 103, 14.9 103, 23.3 103, and 30.3 103 cells/mL. The
mixtures were incubated for about 2 h, and then their absorption spectra were
measured.
2.5. NIR absorption imaging of cancer cells
2.5.1. NIR absorption imaging system
A home-made NIR absorption imaging system was set up in a dark room in order
to prevent stray light. An LED (16 V, 5.0 W, 800 nm 10 nm) serves as the light
source and the image is recorded by a CCD camera (MTV-1881EX, MINTRON)
equipped with a 790e810 nm band-pass filter to eliminate scattering from other
wavelengths. The LED and CCD are placed, respectively, on the bottom and top of a
transparent sample stage in 180 geometry. The system control and image acquisition are via computer software written by ourselves.
2.5.2. Imaging experiments
The CAL-27 cancer cells and primary human oral epithelial cells were used as the
imaging samples. The cells were distributed in 96-well plates with different concentrations of 1 104, 2 104, 3 104, 4 104, and 5 104 cells/well, and incubated
with 0.45 nM RB-GNR probes at 37 C for 2 h. The probe targeted cells were allowed
to attach to the plate, and the cells were rinsed twice with PBS to remove the unbound probes, 200 mL of water were added to increase the optical path. The 96-well
plate was then examined by using the NIR absorption imaging system described
above, and the water sample was used as the basement. Under software control, the
NIR absorption data were acquired and transferred to the output pseudo-color
images, and data representing “LSPR absorption intensity” of the samples.
3. Results and discussion
3.1. Preparation and characterization of RB-GNR nanoprobes
In this work, RB is used as the specific agent and conjugated on
GNRs to obtain the RB-GNR probes (RB-GNRs) for oral cancer diagnostics. The TEM image of the synthesized RB-GNRs is shown in
Fig. 1. It can be seen that the average diameter and length of the RBGNRs are 13 2 nm and 52 4 nm, respectively. However, their
average hydrodynamic diameter is about 60 nm (inset in Fig. 1)
which is larger than the dimensions measured by TEM possibly due
to the contributions from the polyelectrolyte and RB coating. The
results demonstrate that the RB-GNRs have the suitable size and
good dispersibility in an aqueous solution.
Fig. 2 schematically illustrates the preparation of the RB-GNR
probes. The original CTAB-coated GNRs are synthesized by using
a well known seed-mediated growth method utilizing the CTAB
Fig. 1. TEM image and hydrodynamic distribution (inset) of RB-GNRs.
Fig. 2. Schematic illustration of the synthesis of RB-GNRs.
surfactant [24]. A cationic polyelectrolyte (PAH) is used to modify
the surface of the GNRs to obtain PAH-coated GNRs (PAH-GNRs).
The PAH-GNRs were mixed with RB molecules in an aqueous solution for 3 h to facilitate absorption of RB on the GNR surface. After
the PAH-GNRs þ RB solution is centrifuged, the supernatant containing excess RB is removed, and the RB-GNRs in the precipitate is
collected and re-dispersed in an aqueous solution to obtain the RBGNRs. To demonstrate the effects of PAH in the synthesis of the RBGNRs, a control experiment in which the original GNRs are directly
mixed with RB without PAH modification is performed. The
GNRs þ RB solution is centrifuged and the supernatant and precipitate were collected.
Fig. 3a and b show the absorption spectra of the samples prepared in the GNRs þ RB and PAH-GNRs þ RB experiments. As
shown in Fig. 3a, the characteristic RB absorption bands at 510 and
551 nm are observed from the GNRs þ RB solution before centrifugation. However, no obvious RB absorption peaks can be found
from the precipitate of the GNRs þ RB solution after centrifugation,
demonstrating that little or no RB is conjugated to the GNRs
without PAH modification.
In contrast, in the PAH-GNRs þ RB experiment, RB absorption
bands can be observed from both the PAH-GNRs þ RB solution and
precipitate from the PAH-GNRs þ RB solution after centrifugation
(see Fig. 3b). The results clearly indicate RB is coated on the PAHGNRs. Compared to the PAH-GNRs and pure RB, the TSPR peak of
the RB-GNRs blue-shifts slightly and the RB absorption band redshifts slightly. This is induced by plasmonic-molecular resonance
coupling in which molecules near the plasmonic nanoparticles
exhibit light absorption around the plasmon resonance peak [55].
Therefore, such plasmonic-molecular resonance coupling confirms
successful coating of RB molecules on the PAH-GNRs.
The absorption and fluorescence spectra of the supernatants in
the GNRs þ RB and PAH-GNRs þ RB experiments are depicted in
Fig. 3c and d. Compared to those acquired from the original RB, the
absorption and fluorescence intensity of RB in the supernatant of
the PAH-GNRs þ RB solution after centrifugation decreases by 81%
and 79%, respectively. Most of the RB is absorbed by the PAH-GNRs
and only a small amount of RB residues can be found in the supernatant. In contrast, in the GNRs þ RB experiment, the absorption
and fluorescence intensity of RB respectively decreases by only 14%
and 15%, respectively, providing evidence that only a small amount
of RB is coated on the GNRs without PAH modification [54]. The
results further confirm that the RB has been successful conjugated
onto the PAH-GNRs via the interaction between the RB and PAH.
Furthermore, by estimating the amount of RB absorbing on the
PAH-GNRs, the number of RB molecules attached to each single
GNR is determined to be about 2000.
J.-H. Wang et al. / Biomaterials 34 (2013) 4274e4283
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Fig. 3. Comparison of the optical properties between the samples obtained in the GNRs þ RB and PAH-GNRs þ RB experiments to elucidate the RB conjugation mechanism: (a)
Absorption spectra of original CTAB-coated GNRs (black), GNRs þ RB solution before centrifugation (red), and precipitate from the GNRs þ RB solution after centrifugation (blue). (b)
Absorption spectra of PAH-GNRs (black), PAH-GNRs þ RB solution before centrifugation (red), precipitate from the PAH-GNRs þ RB solution after centrifugation (blue), and pure RB
(orange). (c, d) Absorption spectra (c) and fluorescence spectra (d) of original RB (black), supernatant of GNRs þ RB solution after centrifugation (red), and supernatant of PAHGNRs þ RB solution after centrifugation (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The zeta potential is measured to determine the surface charge
of GNRs in the aqueous solution (Fig. 4). The zeta potential of the
original CTAB-coated GNRs is 43.9 mV. After modification with
positively-charged PAH, the zeta potential of the PAH-GNRs increases to 56.0 mV. After negatively-charged RB is conjugated to
the GNRs, the zeta potential of the RB-GNRs declines to 27.3 mV.
The zeta potential of RB-GNRs is still positive but close to zero,
demonstrating that the GNRs are covered by RB partly and corresponding to higher cell transmission than PAH-GNRs [56].
According to the above results, the mechanism of the conjugation between RB and GNRs can be concluded as follows. Owing to
the negatively-charged carboxylic group (COO) and aromatic
hydroxyl group ((Ar)ReO) of RB (see Fig. 2), the RB molecules can
conjugate with the positive-charged GNRs via electrostatic interaction [57]. Therefore, the RB can be conjugated to the original
CTAB-coated GNRs (see Fig. 3c). Similarly, the RB can also be conjugated to the positively-charged PAH-GNRs by electrostatic interaction. However, more RB molecules are conjugated to the PAHGNRs than original CTAB-coated GNRs with the similar zeta potential (see Fig. 3), suggesting that there is another interaction on
the PAH-GNRs. Considering the carboxylic group (COO) of RB
and amine groups (eNHþ
3 ) of PAH, RB can also be bound to PAHGNRs by covalent bonds generated in the condensation reaction.
These two processes constitute the mechanism of the conjugation
of RB to PAH-GNRs to finally obtain the RB-GNR probes.
The stability of the RB-GNR probes in the aqueous solution is
examined (see Fig. 5). Fig. 5a shows the absorption spectra of the
RB-GNRs dissolved in the aqueous solution for different time at
room temperature (w25 C). After standing for one week, both the
GNR LSPR band and RB absorption band of the probes are almost
Fig. 4. Zeta potentials: (1) original CTAB-coated GNRs, (2) PAH-coated GNRs, and (3)
RB-GNRs.
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Fig. 6. MTT assays showing the cytotoxicity of RB-GNR probes.
3.2. SPR sensing assay for oral cancer cells
3.2.1. Sensing mechanism
The SPR bands of the GNRs result from the collective oscillation
of surface electrons and so the resonance frequency of the SPR
bands depends strongly on the dielectric properties, local environment, and aggregation of GNRs [34]. Furthermore, as a specific
agent of oral cancer cells, the RB molecules on the GNRs conjugate
with the protein or nucleic acid from the cancer cell lysate to
Fig. 5. (a) Absorption spectra of RB-GNRs in the aqueous solution after different
standing time at room temperature (w25 C) and (b) absorption spectra of RB-GNRs in
the aqueous solution with and without FBS.
unchanged, demonstrating good stability of the RB-GNR probes in
the aqueous solution. After standing for three weeks, a little redshift in the LSPR band can be found and it is probably due to tiny
aggregation of the probes. Fetal bovine serum (FBS) is used as a
competitive protein to further assess the stability of conjugation
between the RB and PAH to the GNRs. Fig. 5b shows the absorption
spectra of the RB-GNR probes dissolved in aqueous solutions with
0%, 1%, and 10% FBS and kept at 37 C for 4 h. Although a small tail is
found at long wavelengths, no obvious change is observed from
both the LSPR peak wavelength and RB absorption band, demonstrating little influence from the FBS protein on the RB conjugation.
The increased UV absorption is ascribed to the larger FBS content.
All in all, the experimental results demonstrate the RB-GNR probes
have good and long-lasting stability even in a complex environment with high protein concentrations.
The cytotoxicity results are shown in Fig. 6. The cell viability
declines as the concentration of RB-GNRs increases. It may be
caused by a rise in the free RB on the probes or change in the
intracellular osmotic pressure. The high viability values determined
from the cells indicate that even a high sample concentration of
0.45 nM does not generate much cytotoxicity. Such concentration is
much higher than the highest concentration used below for cell
sensing and imaging.
Fig. 7. Sensing assay for oral cancer cells utilizing RB-GNRs as the sensing probes. (a)
Schematic representation of mechanism of SPR sensing. (b) Absorption spectra of RBGNR probes before (black) and after (red) treated with CAL-27 cancer cell lysate. The
inset TEM image shows the assembled RB-GNRs induced by the cancer cell lysate. (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
J.-H. Wang et al. / Biomaterials 34 (2013) 4274e4283
inducing aggregation of the RB-GNR probes. Based on this mechanism, the immunoassay for detection of oral cancer cells based on
the RB-GNR platform is established. The assay is label free and the
experimental protocol is very simple. As shown in Fig. 7a, a certain
volume of the cell lysate aqueous solution is added to the RB-GNR
solution. The mixture is incubated for 2 h and the absorption
spectra are acquired on a UVeViseNIR spectrophotometer. An
obvious red-shift is observed from the LSPR peak of the mixture
containing the RB-GNRs and cancer cell lysate, as shown in Fig. 7b.
The inset TEM image in Fig. 7b suggests that the red-shift can be
ascribed to the end-to-end assembly of the RB-GNRs thereby confirming the specificity of the RB molecules to oral cancer cells. The
results also indicate that the LSPR peak wavelength is a sensitive
parameter to monitor target specific binding of the RB-GNRs onto
the cancer cells. The red-shift in the LSPR peak is much bigger than
that of the TSPR peak, suggesting that the assay based on the LSPR
of the GNRs has higher sensitivity than traditional sensing assays
based on TSPR of gold nanoparticles.
3.2.2. Specificity
The specificity of the sensing assay is assessed. Two sets of RB-GNRs
are treated with the same amount of the lysate from the cancer cells
and normal cells, respectively. As shown in Fig. 8a and b, the LSPR peak
of the RB-GNR probes display an obvious red-shift of w32 nm after
reacting with the lysate of cancer cells, but only a small red-shift of
w7 nm is observed in the case of oral normal cells (see Fig. 8b). The
large difference in the red-shifts demonstrates the specificity of the
sensing assay to cancer cells. The specificity of the RB-GNR probes is
further evaluated using the CTAB-coated GNRs and PAH-coated GNRs
as control samples. As shown in Fig. 8c and d, almost no red-shift is
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observed from the two control samples after treatment with the lysate
of cancer cells. As it is known that RB has high specificity to oral cancer
[41], and has been used as an effective agent for detection of oral
precancerous and malignant lesions with colorimetric evaluation [44].
Here, the RB-GNR probes have specificity in cancer cell sensing
attributable to the specificity of the coated RB molecules.
3.2.3. Quantitative sensing
Quantitative sensing experiments are conducted on two cancer
cell lines of CAL-27 and Tca8113 at different concentrations of cell
lysates, and the cancer cell debris with cell membranes and organelle
membranes serve as the control. As shown in Fig. 9a and b, a much
larger red-shift is observed from the LSPR peak for the cancer cell
lysate than the cell debris. The red-shifts also increase linearly with
increasing cancer cell concentrations in the range from 2.2 103 to
30.3 103 cells/mL. The detection limit defined here as the lowest
concentration in the sensitivity regime is about 2000 cells/mL. In
contrast, no significant signal appears from the control samples (cell
debris), verifying that the effect of nonspecific interactions on the
RB-GNRs is minimal. Similar results are obtained from the other oral
cancer cell line Tca8113, thus furnishing strong evidence that
this sensing method is suitable for different oral cancer lines. All in
all, this label-free sensing platform based on RB-GNR probes is
specific, sensitive, non-invasive, economical, and convenient
and suitable for quantitative optical diagnostics of oral cancer cells.
3.3. NIR absorption imaging assay for oral cancer cells
3.3.1. Principle of NIR absorption imaging system
NIR light penetrates deeply into cells and tissues. The strong
optical absorption by the RB-GNRs in the NIR region enables not
Fig. 8. Specificity of the SPR sensing assay. (a, b) Absorption spectra of 4.5 mL of 0.45 nM RB-GNR probes before (black) and after (red) treatment with 60 mL of (a) CAL-27 oral cancer
cell lysate and (b) oral normal cell lysate. (c, d) Absorption spectra of 4.5 mL of 0.45 nM (c) CTAB-coated GNRs and (d) PAH-coated GNRs before (black) and after (red) treatment with
60 mL CAL-27 cancer cell lysate. The cell density of the cell lysate is 1 106 cells/mL. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
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J.-H. Wang et al. / Biomaterials 34 (2013) 4274e4283
Fig. 9. Quantitative detection of oral cancer cells. (a) Absorption spectra of RB-GNRs reacted with increased concentrations of CAL-27 cell lysate (left) and cell debris (right). (b) Redshifts in the LSPR peak as a function of concentration of CAL-27 cell lysate (red) and cell debris (black). (c) Absorption spectra of RB-GNRs after reacting with increased concentrations of Tca8113 cell lysate (left) and cell debris (right). (d) Red-shifts in the LSPR peak as a function of concentrations of Tca8113 cell lysate (red) and debris (black). (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
only SPR sensing but also absorption imaging [39]. In our experiments, an NIR-optical absorption imaging system is established to
perform the bio-imaging. As shown in Fig. 10a, an LED serves as the
light source and the image is recorded by a CCD camera equipped
with a 790e810 nm band-pass filter. The LED and CCD are placed,
respectively, on the bottom and top of a transparent sample stage in
180 geometry. The system control and image acquisition are via
computer software written by ourselves.
As shown in Fig. 10b, the pseudo-color images in the inset obtained by the system reveal NIR absorption signals (790e810 nm)
from the RB-GNR probes at different concentrations. The sum of
the digital color value of all pixels is calculated to represent the
LSPR absorption intensity in the quantitative analysis. As shown in
Fig. 10b, the calculated the LSPR absorption intensity is proportional to the concentration of the RB-GNR probes between 5 and
40 pM. Comparing the data with those obtained by spectrophotometer (Figs. S2a and S2b), it can be concluded that the NIR absorption imaging system is capable of indicating the amount of
GNR-targeted samples.
3.3.2. NIR absorption imaging assay for oral cancer cells
Based on the imaging system described in the previous section,
an imaging assay is established to detect oral cancer cells. As shown
in Fig. 11a, different amounts of CAL-27 cancer cells and normal
cells are cultured on a 96-well plate with the medium containing a
certain amount of the RB-GNR probes with an overdose (final
concentration ¼ 0.45 nM) for 2 h. The samples were rinsed with PBS
twice to remove the unbound probes and 200 mL of ultrapure water
were added. Considering the specificity of the probes to the cancer
cells, the absorption intensity of the bounded RB-GNR probes can
be used to indicate the amount of the cancer cells. Fig. 11b shows
the pseudo-color images obtained from the samples containing
different amounts of the cancer cells. It can be seen that the absorption intensity increases obviously with cancer cell quantities,
while much weaker absorption signals are obtained in the normal
cells, demonstrating the specificity of the imaging assay for oral
cancer cells. In addition, Fig. 11c reveals a linear correlation between the LSPR absorption intensity I790-810 and the amount of
cancer cells “C” between 10 and 50 103 cells/mL I790810 ¼ 0.58C þ 29.5. The results demonstrate that the imaging assay
containing the RB-GNR probes can detect cancer cells quantitatively. It should also be noted that these ten samples of cells are
assessed simultaneously, indicating that it is a multi-channel
detection method in which multiple samples can be analyzed at
the same time. Hence, this method is particularly useful for rapid
and high-throughput early cancer screening.
J.-H. Wang et al. / Biomaterials 34 (2013) 4274e4283
4281
Fig. 10. (a) Schematic of the NIR-optical absorption imaging system. (b) Absorption
intensity as a function of RB-GNR probe concentration in aqueous solutions, with the
pseudo-color images in the inset showing the concentration-dependent NIR absorption images of the RB-GNR probes in the aqueous solutions.
Fig. 11. (a) Schematic presentation of the detection process of the NIR absorption
imaging assay for oral cancer cells. (b) Pseudo-color images showing amountdependent NIR absorption images of cancer cells (up) and normal cells (down). (c)
LSPR absorption intensity I790e810 as a function of amount of cancer cells and normal
cells in aqueous solutions.
3.4. Discussion
are label-free and convenient and are thus more efficient for
screening examinations of oral cancer. Secondly, fluorescence
spectroscopy which is the most commonly used detection technique in immunoassays requires moderately expensive instruments, and fluorophores are easily susceptible to
photodestruction. In contrast, our assays are based on the NIRoptical absorption signals from the GNRs. Such metal nanomaterials are not prone to photodestruction, and the instruments
required are much cheaper.
The above results show the sensing and imaging assays
employing the RB-GNR platform for the optical diagnostics of
oral cancer. Both of these two assays are based on the NIR absorption signal from the RB-GNR probes, however have different
detection objects and are according to distinct mechanism. The
sensing assay uses the red-shifted wavelength in the LSPR absorption band of the probes to indicate the amount of the cancer
cell lysate in the aqueous solution; while the imaging assay use
the LSPR absorption intensity of the probes to indicate the
amount the cancer cells. The results indicate that these two assays are specific and quantitative. The sensing assay is more
sensitive and suitable for accurate optical diagnostics, whereas
the imaging approach which offers multi-channel and rapid
detection is more suitable for screening examinations of a large
number of cases.
The advantages of the established assays are further demonstrated by comparing to traditional diagnostics methods. Firstly, it
is known that the currently used non-invasive cancer diagnosis
techniques include ELISA, micro-satellite analysis, and high performance liquid chromatography (HPLC), all of which need laborintensive procedures. In contrast, the two assays described here
4. Conclusion
RB-GNRs are produced synthesized by conjugating RB molecules and GNRs using a special technique. The materials exhibit
good dispensability, low cytotoxicity, strong LSPR absorption band
in the NIR region, and specificity to oral cancer cells. Oral cancer
cells are tested using the RB-GNR platform which can be used as
both a sensor and imaging probe. The sensing assay is more sensitive and accurate, whereas the imaging approach offers multichannel and rapid detection. Both modes are highly-efficient, and
convenient, and this RB-GNR platform has immense potential in
cost-effective diagnostics of oral cancer.
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J.-H. Wang et al. / Biomaterials 34 (2013) 4274e4283
Acknowledgments
The authors thank Dr. Zhonghua Hao and Mr. Min Luo in Wuhan
University for technique help. The authors acknowledge financial
support from the Natural Science Foundation of China (10874134
and 10904119), the Natural Science Foundation of Jiangsu Province
(SBK201240182), as well as Hong Kong Research Grants Council
(RGC) General Research Funds (GRF) Nos. CityU 112510 and 112212.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2013.02.012.
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