Platelet-derived growth factor oncoprotein detection using three-dimensional carbon microarrays.
ABSTRACT The potential of aptamers as ligand binding molecule has opened new avenues in the development of biosensors for cancer oncoproteins. In this paper, a label-free detection strategy using signaling aptamer/protein binding complex for platelet-derived growth factor (PDGF-BB) oncoprotein detection is reported. The detection mechanism is based on the release of fluorophore (TOTO intercalating dye) from the target binding aptamer's stem structure when it captures PDGF. Amino-terminated three-dimensional carbon microarrays fabricated by pyrolyzing patterned photoresist were used as a detection platform. The sensor showed near linear relationship between the relative fluorescence difference and protein concentration even in the sub-nanomolar range with an excellent detection limit of 5pmol. This detection strategy is promising in a wide range of applications in the detection of cancer biomarkers and other proteins.
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ABSTRACT: A novel method for the detection of PDGF-BB has been developed using double-strand DNA-copper nanoparticles (dsDNA-CuNPs) as fluorescent markers. This assay relies on the premise that the aptamer-based probe undergoes a conformational change upon binding with target protein, and subsequently triggers polymerization reaction to generate dsDNA. Then, the resultant dsDNA can be used as a template for the formation of CuNPs with high fluorescence. Under the optimized conditions, the proposed assay allowed sensitive and selective detection of PDGF-BB with a detection limit of 4 nmol/L. This possibly makes it an attractive platform for the detection of a variety of biomolecules whose aptamers undergo similar conformational change.Chinese Chemical Letters 01/2013; · 1.18 Impact Factor
Platelet-derived growth factor oncoprotein detection using
three-dimensional carbon microarrays
Varun Penmatsaa, A. Rahim Ruslindab,c, Majid Beidaghia, Hiroshi Kawaradab, Chunlei Wanga,n
aDepartment of Mechanical and Materials Engineering, Florida International University, United States
bSchool of Science and Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku, 169-8555 Tokyo, Japan
cInstitute of Nano Electronic Engineering (INEE), Universiti Malaysia Perlis, Jln seriab?Alor setar, 01000 Kangar, Perlis, Malaysia
a r t i c l e i n f o
Received 6 April 2012
Received in revised form
13 June 2012
Accepted 29 June 2012
Available online 20 July 2012
3D carbon microarrays
Platelet-derived growth factor
a b s t r a c t
The potential of aptamers as ligand binding molecule has opened new avenues in the development of
biosensors for cancer oncoproteins. In this paper, a label-free detection strategy using signaling
aptamer/protein binding complex for platelet-derived growth factor (PDGF-BB) oncoprotein detection
is reported. The detection mechanism is based on the release of fluorophore (TOTO intercalating dye)
from the target binding aptamer’s stem structure when it captures PDGF. Amino-terminated three-
dimensional carbon microarrays fabricated by pyrolyzing patterned photoresist were used as a
detection platform. The sensor showed near linear relationship between the relative fluorescence
difference and protein concentration even in the sub-nanomolar range with an excellent detection limit
of 5 pmol. This detection strategy is promising in a wide range of applications in the detection of cancer
biomarkers and other proteins.
& 2012 Elsevier B.V. All rights reserved.
With the increasing application of proteomic strategies for the
detection of cancer related oncoproteins and discovery of bio-
markers, it is of extreme interest to develop portable platforms for
sensitive detection of proteins and their molecular variants.
Aptamers are single stranded DNA or RNA molecules selected
in vitro from DNA/RNA random pools that are capable of binding
with biological entities such as proteins, cells along with small
molecules, drugs, peptides and hormones with high affinity and
specificity (Ellington and Szostak, 1990; Robertson and Joyce,
1990; Tuerk and Gold, 1990). Aptamers have been sought out as
ideal alternative candidates to the traditional antibodies for use in
analytical devices due to their easy synthesis, high binding
affinity, long storage times, and excellent selectivity (Jayasena,
1999). Recent studies have demonstrated the applicability of
aptamers to target a disease state, such as cancer (Shangguan
et al., 2006). This opens up new avenues in the future for
aptamers to potentially substitute more established components
for therapeutics and/or diagnostics.
Platelet-derived growth factor (PDGF) is a protein that regu-
lates cell growth and division. Overexpression of PDGF has been
associated with several human health disorders including athero-
sclerosis (hardening of the arteries) (Lassila et al., 2004), balloon
injury induced restenosis (narrowing of blood vessels) (Szabo ´
et al., 2007), pulmonary hypertension (Barst, 2005), organ fibrosis
(formation of excess fibrous connective tissue in an organ or
tissue) (Trojanowska, 2008), tumorigenesis (formation of tumors)
(Shih et al., 2004). PDGF receptors are almost undetectable in
normal vessels, but are highly expressed in the diseased vessels. A
PDGF dimer composed of two different types of monomer (A and
B chains) occurs in three variants: PDGF-BB, PDGF-AB and PDGF-
AA. In particular, oncoprotein PDGF-BB is often overexpressed in
human malignant tumors and known as a potential protein
marker for cancer diagnosis (Shih et al., 2004).
In recent years, PDGF-BB protein detection using fluorescence
(Yang et al., 2005, 2007; Ruslinda et al., 2012; Fang et al., 2001, 2003;
Vicens et al., 2005; Jiang et al., 2004; Zhou et al., 2006; Huang et al.,
2007, 2008) colorimetry (Huang et al., 2005) and electrochemistry
techniques have been reported (Lai et al., 2007; Degefa and Kwak,
2008; Ruslinda et al., 2010). These methods involve either labeling
the aptamer with a fluorophore, or the use of redox species. In
fluorescence based PDGF detection techniques, fluorophore-labeled
aptamers are used to signal binding by monitoring the changes of
fluorescence intensity (Fang et al., 2003) or anisotropy resulting from
the changes of the microenvironment (Fang et al., 2001) or rotational
motion through fluorescence energy transfer (Vicens et al., 2005).
However, as the precise target binding sites and the conformational
changes of the aptamers are generally unknown, it is not easy to
design labeling strategies. Besides, there is always a concern that the
conjugation of a fluorophore to an aptamer will ultimately weaken
the affinity of the aptamer to its ligand (Huang et al., 2007). In the
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/bios
Biosensors and Bioelectronics
0956-5663/$-see front matter & 2012 Elsevier B.V. All rights reserved.
nCorresponding author. Tel.: þ1 305 348 1217.
E-mail address: firstname.lastname@example.org (C. Wang).
Biosensors and Bioelectronics 39 (2013) 118–123
case of electrochemistry based detection techniques, due to the use of
redox species, the electrodes are limited to conductive materials and
also the different linkers used to attach the aptamer onto the
electrode surface (such as gold) exhibits rapid degradation with time
(Phillips et al., 2008). Most recently, diamond substrate has been used
to detect PDGF by monitoring the fluorescence change from the
release of an intercalating dye when the probe aptamer captures the
target (Ishii et al., 2011). Although the sensor showed good sensitivity
and selectivity, the use of diamond substrates is not cost effective. The
controllability of defects and grain boundaries in polycrystalline
diamond substrates along with the high operating cost due to the
need for high vacuum and high temperature systems are limiting
factors for mass production.
Traditionally for biological and electrochemical sensing, glassy
carbon is one of the popularly used materials due to its low cost,
better resistance towards biofouling, biocompatibility, good electrical
conductivity, low background capacitance, and the flexibility to tailor
the surface by various physical/chemical treatments. In particular,
carbon synthesized by carbon-microelectromechanical systems (C-
MEMS) technique (also known as pyrolyzed photoresist carbon),
where organic photoresist patterns are heat treated at high tempera-
tures and oxygen free environment, is intriguing since it exhibits
reaction kinetics comparable to glassy carbon, but with lower
oxygen/carbon atomic (O/C) ratio (Wang et al., 2005; Ranganathan
et al., 2000, Singh et al., 2000). Since photolithography technique is
used for patterning purpose, the electrodes obtained by this manner
have better resolution and reproducibility compared to screen printed
carbon paste electrodes. C-MEMS technique is actively pursued to
fabricate electrodes for DNA biosensors (Yang et al., 2009), glucose
sensors (Xu et al., 2008), protein detection (Lee et al., 2008),
microbatteries (Wang et al., 2004) and on-chip supercapacitors
(Chen et al., 2010) due to the versatility in the experimental approach
to produce high surface area 3D carbon microarrays. In addition, our
group has already demonstrated the ability to tailor the carbon
surface by introducing nanoporosity using a block copolymer as
porogen (Penmatsa et al., 2010) and integration of functional nano-
materials such as graphene (Penmatsa et al., 2012a) and carbon
nanotubes on the surface of 3D carbon microarrays (Chen et al.,
2010). The high surface area of the 3D carbon microarrays makes it an
ideal platform for increased biomolecule loading to improve the
sensitivity and performance of the functional devices. In addition,
compared to fluorescence detection on 2D carbon micropatterns
fabricated on SiO2/Si substrate, 3D micropillar arrays does not suffer
from optical interferences obtained on oxidized silicon substrates.
This phenomena of modulating fluorescence intensity by using
optical interferences leads to strong fluorescence from unbinded
flouropore on the SiO2/Si substrate (Bras et al., 2004; Oillic et al.,
2007; Lambacher and Fromherz, 1996).
In this work, we report a signaling aptamer/protein binding
complex on 3D carbon micropillar arrays using TOTO intercalating
dye to signal PDGF-BB–aptamer binding. The carbon surface was
functionalized by direct amination technique to introduce amino
groups for covalent immobilization of target binding aptamer. It was
demonstrated that this simple detection technique offered high
sensitivity with PDGF detection in the sub-nanomolar range and
good selectivity against different proteins, which could be extended
for the detection of other biomarker proteins.
2.1. Fabrication of 3D carbon microarrays
The three-dimensional carbon microarrays were fabricated by
a typical C-MEMS process. 4 in. silicon oxide wafers were spin
cleaned by acetone and methanol followed by a 5 min bake on the
hotplate at 150 1C for 5 min to evaporate any moisture. NANOTM
SU-8 100 negative photoresist was spin coated using a photoresist
spinner (Headway researchTM) at 500 rpm for 12 s and then
1200 rpm for 30 s to get approximately 200 mm photoresist films.
The photoresist was baked at 65 1C for 10 min and at 95 1C for
30 min in order to harden the photoresist by evaporating any
remaining solvents. The photoresist was patterned by exposure
using OAI Hybralign contact aligner (light intensity, 17 mW/cm2)
for 60 s to crosslink polymer chains in the photoresist. Post-
expose bake was carried out at temperatures of 65 1C for 1 min
and 95 1C for 3 min respectively to further harden the crosslinked
NANOTMSU-8 developer (Microchem, USA) for 15 min to wash
away the unwanted photoresist. The pyrolysis of the photoresist
microarrays was conducted in a Lindberg tube furnace under (95%
N2þ5% H2) environment. The samples were heated from room
temperature to 350 1C at 2 1C/min rate with a hold time of 40 min,
followed by ramping to 1000 1C at 5 1C/min rate and hold time of
60 min. The samples were cooled down to room temperature in
the inert atmosphere
2.2. Surface functionalization using direct amination technique
Before the direct amination process, the samples were first
thoroughly rinsed with DI water and blow dried. The amination
process was performed at room temperature in an ammonia gas
253.7 nm). Prior to UV irradiation, the reaction chamber was
purged with nitrogen gas for 5 min to remove oxygen and other
gases. The reaction chamber was then irradiated with UV light for
4 h under a continuous flow of ammonia gas at 100 sccm. Finally,
nitrogen gas is purged for 5 min to remove any ammonia in the
reaction chamber before removing the sample.
2.3. PDGF detection
The 50-carboxyl-modified PDGF-B-binding aptamer (50-CAG GCT
ACG GCA CGT AGA GCA TCA CCA TGA TCC TG-30), PDGF-BB, PDGF-AB,
PDGF-AA, adenosine triphosphate (ATP), and calmodulin were pur-
chased from Sigma Genosys. The intercalating dye 1,1-(4,4,8,8-
tetra(benzo-1,3-thiazole)-2-methylidene] (quinolinium tetraio-
dide (TOTO)) was purchased from Invitrogen Corporation. The
carboxyl modified aptamer was covalently immobilized on the
amino- terminated carbon surface without the use of any linker
molecules. The probe aptamer with 3?sodium saline citrate
(SSC) buffer solution, 0.1 M N-hydroxysuccinimide (NHS) and
0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDC) were mixed in a 2:1:1 ratio. The final concentra-
tion of the probe aptamer solution was 20 mM. 5 ml of the probe
aptamer solution was dropped onto the 3D carbon microarrays
and incubated for 2 h at 38 1C in a humidified chamber. After
immobilization, the sample was washed in PBSþTween-20 (PBS:
1 mM NaCl: 2 mM NaH2PO4: 8 mM Na2HPO4: 0.1% Tween-20)
solution for 5 min and three times with deionized (DI) water for
3 min each The probe aptamer was then reacted with 10 mM
intercalating dye (TOTO) diluted in TE buffer [10 mM tris(hydrox-
ymethyl)-aminomethane (Tris), 1 mM ethylenediaminetetraace-
intercalation of the dye, the sample was cleaned by TE buffer
for 20 min and a DI water rinse. PDGF-BB protein diluted in
2?SSC was then bound to the immobilized aptamer at room
temperature for 1 h at 25 1C. Unbound PDGF-BB were cleaned by
DI water for 5 min. It is noteworthy that certain monovalent and
divalent cations commonly encountered in biological specimens
are known to affect DNA conformation. For this reason, we
selected the concentrations of the solution based on our previous
for1 hat25 1C.Followingthe
V. Penmatsa et al. / Biosensors and Bioelectronics 39 (2013) 118–123
study concerning the effect of protein binding based on Mg2þ
cation and NaCl concentration in PBS buffer solution. Finally, in
order to regenerate the sensor by dissociating PDGF-BB and
intercalator from the probe aptamer, the sample is washed in
10% sodium dodecyl sulfate (SDS) solution for 30 min.
The morphology of 3D carbon microarrays was investigated
using JOEL 6335 FE-SEM scanning electron microscopy. Raman
spectrum was collected with an argon ion laser system (Spectra
Physics, model 177G02) of l¼514.5 nm at a laser power of ca.
7 mW. The chemical composition of pyrolyzed photoresist carbon
film before and after direct amination procedure was investigated
by an Ulvac F 3300 X-ray photoelectron spectroscopy (XPS) with
an anode source providing Al Ka radiation. The electron takeoff
angle was 45731 relative to the substrate surface. Fluorescence
observation was performed using an Olympus IX71 epifluores-
3. Results and discussion
A typical SEM image of high aspect ratio 3D carbon micropillar
arrays is shown in Fig. 1a. The average dimensions of the carbon
micropillars after carbonizing patterned SU-8 photoresist struc-
tures are ?160 mm height and ?30 mm width. A careful exam-
ination of the SEM image shows that the upper half and specially
the top part of the carbon micropillars is slightly wider compared
to the lower half. This could be due to the higher dose of UV light
experienced by the top layer of the thick photoresist (Wang et al.,
2005). Raman spectroscopy was used to investigate the crystal-
linity of the carbon micropillars. Fig. 1b shows the Raman
spectrum of pyrolyzed carbon with two significant broad peaks
at ?1350 cm?1(D-band) and ?1590 cm?1(G-band). The first
peak at 1350 cm?1represents the disorder band of the micro-
crystallite graphite and the second peak at 1590 cm?1is due to
the single Raman line typically found on single crystalline
graphite. The ID/IG ratio of 1:1 indicates that carbon obtained
from pyrolysis of photoresist is identical to glassy carbon synthe-
sized at same temperature (Penmatsa et al., 2012b).
It is well documented that the termination or functionalization
of the surface is one of the key issues in the interaction and
immobilization of biomolecules (Kawarada and Ruslinda, 2011).
In this work, to covalently immobilize PDGF binding aptamer on
the carbon surface, the sample was first treated by direct amina-
tion technique (Yang et al., 2009) where the sample was irra-
diated by ultraviolet (UV) light (l¼253.7 nm) in an ammonia gas
environment for 4 h. In contrast to oxidation techniques which
introduce several oxygen-based functional groups such as ketone,
hydroxyl, and carboxyl groups, only NH2bonds are expected to
form on the carbon surface by direct amination procedure due to
their chemical structure. The elemental composition and surface
binding of pyrolyzed photoresist film were evaluated by X-ray
photoelectron spectroscopy spectra (XPS) as shown in Fig. 2.
Analysis of the widescan XPS spectra of the bare carbon film
before amination (Fig. 2 inset) shows two major peaks evident of
carbon (284.6 eV) and oxygen (531.8 eV) but in the case of after
amination, three distinct peaks representing carbon, oxygen and
nitrogen (398.4 eV) are evident. The nitrogen peak visible after
amination is a result of ammonia gas forming C–NH2 on the
carbon substrate. The deconvoluted high resolution C1s spectrum
(Fig. 2) shows major carbon peaks at 284.6 eV (sp2) and 285.2 eV
(sp3). The other peaks at 285.4, 286.3, 287.6 and 289.1 eV
corresponds to C–N, C–O, CQO and O?CQO bonds, respec-
tively. The maximum surface coverage of amino groups achieved
was ?8%, which is similar to the amino coverage previously
reported (Yang et al., 2009). While the amination percentage on
the carbon surface was characterized by XPS, characterizing the
active aptamers on the electrode surface is one of the challenges
as pointed out by Ishii et al. (2011). In future research, more effort
should be given to quantitatively characterize the active aptamers
on the electrode surface, specially complex 3D electrode surface.
Fig. 1. (a) Typical SEM image of 3D carbon microarrays and (b) Raman spectrum of pyrolyzed photoresist film showing the two prominent bands at 1350 and 1590 cm?1.
Fig. 2. Deconvoluted C1s spectra of pyrolyzed photoresist film after 4 h direct
amination, here dash line shows the original data and solid lines show the fitting
curves. Inset shows the widescan XPS spectra of carbon film before and after
V. Penmatsa et al. / Biosensors and Bioelectronics 39 (2013) 118–123
Fig. 3. Schematic illustration of the detection of PDGF-BB using signaling aptamer/protein binding complex on 3D carbon microarrays platform; (I) covalent
immobilization of PDGF-binding aptamer on partially aminated carbon surface, (II) intercalating the probe aptamer with TOTO fluorescent dye, (III) binding PDGF-BB
to the aptamer-intercalating dye complex, (IV) regenerating the sensor by sodium dodecyl sulfate (SDS) treatment to remove PDGF and release the intercalating dye.
Fig. 4. (a) Relative fluorescence difference response of the sensor to different concentrations of PDGF from 0.005 nM to 100 nM. The concentrations of the aptamer and
intercalating dye were 20 mM and 10 mM respectively. (b) Comparison of relative fluorescence difference of different proteins towards PDGF binding aptamer. The
concentration of the different molecules (PDGF-BB, PDGF-AB, PDGF-AA, BSA, ATP and calmodulin) was 100 nM and concentrations of PDGF-binding aptamer and
intercalating dye were 20 mM and 10 mM, respectively.
V. Penmatsa et al. / Biosensors and Bioelectronics 39 (2013) 118–123
The detection of PDGF-BB using signaling aptamer/protein
binding complex strategy is shown schematically in Fig. 3.
(I) The carboxyl-terminated PDGF-binding aptamer (probe apta-
mer) is first covalently attached to the amine-terminated carbon
surface via amide binding. (II) Subsequently the TOTO dye was
intercalated with PDGF-binding aptamer. The TOTO dye shows no
fluorescence in aqueous solution but exhibits strong fluorescence
when bound to the nonaqueous pocket of the duplex nucleic acid
regions in the aptamer. It is important to note that the fluores-
cence signal from TOTO is dependent on its local environment
and DNA/RNA conformation. (III) When the target PDGF-BB
protein bonds with the aptamer, the induced conformational
change of the aptamer, as well as the blocking of intercalated
TOTO dye results in a significant protein-dependent fluorescence
change. (IV) Finally for regenerating the sensor, the aptamer
intercalating dye complex and PDGF-BB are dissociated by treat-
ment with sodium dodecyl sulfate (SDS).
The relationship of the change in the relative fluorescence
difference with different concentrations of PDGF-BB in 2?SSC
(saline-sodium citrate) solution was evaluated to study the
sensitivity of the sensor. At first, the difference in the fluorescence
intensities is computed from the fluorescence intensity values
obtained after initial TOTO intercalation with the probe aptamer
and then after PDGF-BB binding with the probe aptamer. Finally,
the relative fluorescence difference is calculated by dividing the
value obtained from difference in fluorescence intensities and
initial fluorescence intensity. As expected, analysis of the data in
Fig. 4a shows that the relative fluorescence difference increased
as the concentration of PDGF-BB was increased from 0.005 to
100 nmol. This can be explained by the fact that, as the PDGF-BB
concentration is increased, more intercalator dye is released from
the aptamer which results in a larger difference in the relative
fluorescence. A near linear relationship between the relative
fluorescence difference and the protein concentration was
observed even in the sub-nanomolar range. A low detection limit
of 0.005 nmol was achieved, and indicates that the sensor detec-
tion limit is much below the typical detection range of the PDGF
in clinical samples. The detection limit by other reported apta-
mer-based analytical techniques, for example, is 1 nmol in undi-
luted serum and 0.05 nmol in 50% serum was achieved with
electrochemical detection (Lai et al., 2007), 0.1 nmol using solu-
tion based fluorescent signaling complex of aptamer and TOTO
(Fang et al., 2001), and 2 nmol with fluorescence anisotropy based
detection (Fang et al., 2001). Typical PDGF concentrations of
normal individuals and cancer patients have been found to be
in the sub-nanomolar range: 0.4–0.7 nmol in human blood serum
and 0.008–0.04 nmol in human plasma (Ruslinda et al., 2012).
Therefore, with the excellent sensitivity we achieved, we expect
that this PDGF sensor has the potential to be used in clinical
Fig. 4b shows the selectivity test of the probe aptamer towards
the three variants of PDGF along with other biological compo-
nents such as bovine serum albumin, calmodulin, and ATP, which
are all typically present in the blood. The graph shows that the
relative fluorescence difference for PDGF-BB binding with probe
aptamer was about two times that of PDGF-AB and 10-times that
of PDGF-AA binding with the same probe aptamer. Further,
fluorescence intensity difference for other biomolecules such as
bovine serum album (BSA), ATP and calmodulin was approxi-
mately 70 fold smaller when compared to the value obtained for
PDGF-BB binding. These results could be explained mainly by the
fact that the PDGF-binding aptamer used in this work binds to the
three isoforms of PDGF (PDGF-BB, PDGF-AB, and PDGF-AA) with
different affinities. Since the target binding aptamer has high
specificity toward PDGF-BB, the corresponding reduction in the
fluorescence intensity caused by PDGF-AA was clearly lower due
to the absence of any binding sites on the aptamer towards PDGF-
AA. On the other hand, PDGF-AB protein consists of both A and B
chains meaning only one site that could bind to the aptamer. The
amino acid sequences of PDGF-A is 60% similar to that of PDGF-B.
Therefore, this sensor can detect isoforms with good selectivity. In
the other cases where different biomolecules such as BSA, ATP
and calmodulin are introduced towards the target binding apta-
mer, no significant binding is expected due to the unavailability of
the binding site and therefore no major relative fluorescence
difference was detected. It is noteworthy that although BSA
usually contains a high concentration of proteins, it does not
affect the selectivity of the probe aptamer used. The selectivity of
the sensing platform achieved in this work towards PDGF-BB
compared to other biological components exhibits the promise of
aptamers for cancer biomarker detection. The sensitivity and
selectivity of our sensor platform could be even further improved
when using the high surface area 3D carbon microarrays inte-
grating with functional nanomaterials such as graphene and
carbon nanotubes. However, for this sensing platform to be used
in clinical applications some future work needs to be conducted,
for example: detection of PDGF-BB from a complex sample
containing a mixture of wide-variety of non-specific recombinant
molecules and a robust study on the reusability of our sensor
platform over multiple cycles of detection and regeneration.
In summary, we demonstrated highly sensitive detection of
PDGF using aptamer/protein binding complex on the 3D carbon
microarray platform. For covalent immobilization of the probe
aptamer, the carbon surface was bio-functionalized using direct
amination technique. The sensor showed a near linear relation-
ship towards protein concentration even in the sub-nanomolar
range with very good selectivity towards PDGF-BB. The robust
platform of signaling aptamer/protein binding complex on 3D
carbon microarrays has the ability to detect wide variety of
biomarkers and proteins for potential application in the preli-
minary diagnosis of cancer.
Varun Penmatsa and Majid Beidaghi gratefully acknowledges
the financial support from the DEA and DYF fellowships provided
by FIU graduate school. This project is partially supported by the
National Science Foundation (OISE 0934078 and CMMI 0800525).
The authors would also like to acknowledge Grant-in-Aid from
GCOE Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan and a Grant-in-Aid for Fundamen-
tal Research of Japan Society for the Promotion of Science (JSPS)
(23246069). The authors would like to acknowledge the research
facilities provided at AMERI at FIU and NTRC at Waseda
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