Sensors and Actuators A 216 (2014) 405–416

Contents lists available at ScienceDirect

Sensors and Actuators A: Physical

journal homepage: www.elsevier.com/locate/sna

Discrimination of colorectal cancer cell lines using

microwave biosensors夽
L.Y. Zhang a , C. Bounaix Morand du Puch b , C. Dalmay a , A. Lacroix c , A. Landoulsi a , J. Leroy a ,
C. Mélin c , F. Lalloué c , S. Battu c , C. Lautrette b , S. Giraud b , A. Bessaudou a , P. Blondy a ,
M.O. Jauberteau c , A. Pothier a,∗
a
XLIM – UMR 7252 Université de Limoges-CNRS, Limoges, France
b
Oncomedics, 1 Avenue d’Ester, 87069 Limoges, France
c
Homéostasie cellulaire et Pathologies EA 3842, Limoges University, Limoges, France

a r t i c l e i n f o a b s t r a c t

Article history: This paper illustrates the potential of microwave frequencies for biological analysis and cell discrim-
Received 30 September 2013 ination. Microwave electric fields have the capability to penetrate inside cells and interact with their
Received in revised form 25 February 2014 intracellular content. Hence, measuring cell electrical properties in the Gigahertz frequency spectrum
Accepted 10 March 2014
may allow distinguishing intracellular differences between cells without requiring any labeling or dena-
Available online 4 April 2014
turation. Here, we present how microwave resonators can be used as biosensors to measure individual
cell dielectric permittivity and obtain characteristic electromagnetic signatures as a function of the cell
Keywords:
type considered, in particular their cancer cell stage (i.e. aggressiveness level). Five cell lines, all derived
Colorectal cancer
Biosensors
from low to high grade human colorectal tumors, were cultured on-chip or simply deposited in water
Microwave resonators droplets on microwave biosensors. For this preliminary study, the sensors only operated in air for fre-
High frequency dielectric spectroscopy quencies ranging from 5 GHz to 14 GHz, allowing establishing a representative electromagnetic signature.
Cancer detection During our experiments, sensors were fully dried, and a fixation step allowed the reproducible character-
ization of cells outside their culture medium, in ambient air, while maintaining their intracellular content
unmodified. Results showed significant electromagnetic signature differences between cell lines, espe-
cially between cells with low and high aggressiveness levels. We also show that a correlation might be
envisioned between the measured electromagnetic signatures and the potential aggressiveness stage. At
this level, we stand at the proof-of-concept stage, and a clinical investigation will be required to establish
from several patients both the reliability and the reproducibility of the potentially existing relationship
between electromagnetic signatures identified in this study and actual cancer progression.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction label and, as a consequence, cannot be reused anymore after anal-

In the last years, there has been an increasing interest for the
development of biosensors combining label-free and high sensitiv-
ity detection. These requirements are crucial in many application
domains especially in the biomedical and public health fields.
Among the large range of biosensing methods such as opti-
cal, mechanical, electrical, etc., label-free detection has recently
attracted a lot of research efforts [1–5]. Indeed, labeling cells
presents many drawbacks: (i) cells can be definitively altered by the
夽 Selected Paper based on the paper presented at The 17th International Con-
ference on Solid-State Sensors, Actuators and Microsystems, June 16–20, 2013,
Barcelona, Spain
∗ Corresponding author. Tel.: +33 587506537.
E-mail address: arnaud.pothier@xlim.fr (A. Pothier).
ysis; (ii) labeling steps are generally expensive, time-consuming
and, for some cases, can be very difficult to set related to the bio-
logical target specificities, as it is for example the case for rare or
undifferentiated cells [6].
To develop new label-free detection strategies, highly sensi-
tive biosensors have been increasingly studied thanks to emerging
micro-technologies that allow designing biosensing devices oper-
ating up to the single cell level [7]. Contrary to conventional
biological methods, which require highly concentrate samples for
the analysis, biosensors can operate on a very low density of cells.
This is very attractive in the diagnostic area especially to detect and
study rare and uncommon cells hidden in a large heterogeneous
cell population [8,9]. Applications of such biosensors are poten-
tially large. It is especially true in cancer research, where detecting
malignant cells at an early stage, while the tumor is still small or
as long as cancer cells are only scarce, remains a challenge. Indeed,

http://dx.doi.org/10.1016/j.sna.2014.03.022
0924-4247/© 2014 Elsevier B.V. All rights reserved.
406 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416
the early diagnosis of cancer is crucial for cancer prevention and
for significantly decreasing the associated mortality rates [10], par-
ticularly for cancers that currently present a low recovery rate or
affect a large number of people every year. In this context, this work
focuses on colorectal cancer, which is the third most common can-
cer worldwide and unfortunately causes an estimated one million
deaths annually [11].
Conventionally, the cancer diagnosis is based on the staging
of tumors. The defined stages range from 0 to IV depending on
the properties of cells that make up the tumor. When the stage
increases, the malignant cells are more aggressive, have a propen-
sity to proliferate and can produce metastases in remote tissues. At
this stage, the efficiency of available treatments generally decreases
significantly and the chances of recovery are very low.
Up to now, biologists have no specific label available to accu-
rately determine the tumor stage. The diagnosis is based on
macroscopic and microscopic observations of tissues and/or cells
from tumor biopsies. Tumor staging systems classify the micro-
scopic cell appearance abnormalities according to changes in
morphology, increase in cell proliferation index or nucleus size.
This method is efficient but presents some disadvantages: (i) it is
complex and invasive since a biopsy or a resection is required; (ii)
it is not an automated process and only a few sections (thin slices)
of the suspicious tissue are observed which lacks sensitivity due to
visual-only observation; (iii) finally, this is a relatively expensive
method which requires time and double reading of tissue slides by
two pathologists in order to limit errors.
That is why an alternative and automated process would be of
great interest to help pathologists to complete their diagnosis. The
discrimination between different stages of tumor would be based
for example on detection of very aggressive cells present inside the
tumor. Such cells should present intracellular content differences
(i.e. change in ionic composition, water proportion, shape and size
of nucleus, etc.) compared to normal or less active malignant cells.
Hence one can notice that a highly sensitive detection method is
required to achieve such discrimination between cells.
In this context, considering that the cell plasma membrane is
transparent at microwave frequencies [12], it seems particularly
pertinent to use such electric fields, in order to probe the cell cyto-
plasm and estimate its dielectric characteristics. It is well known
that changes in a cell, for example from a healthy to a pathological
state, result in variations of its electrical properties. As an exam-
ple, the electrical conductivity and permittivity of normal tissues
are lower than those of cancerous tissues [13,14]. Indeed con-
ductivity and permittivity of biological tissues depend on several
factors: concentration of relevant ions (potassium, sodium, chlo-
ride, magnesium, etc.) [15], proteins and other macromolecules,
water content, nucleus/cytoplasm ratio [16], etc. As a consequence,
tumor stage differences in individual malignant cells may also most
likely result in electrical property differences.
Biological investigations at the tissue scale with microwaves
have been done for several decades [17,18]. Recently some pub-
lished works have demonstrated the possibility to detect and
analyze dielectric properties with RF/microwave-based biosensors
at the cell scale [19–21]. Especially, microwave sensors working
with a resonant principle have been tested exhibiting a superior
sensitivity allowing the single cell detection and analysis [22].
The experiments presented in this paper focus on the microwave
analysis of five colorectal tumor cell lines derived from cancerous
tissues at different stages. For this study, a cell electromagnetic sig-
nature has been either established or initiated for these five cell
populations, using microwave sensors following similar topologies
and design principles than those presented in [23–25]. In this frame,
these cell lines were characterized in ambient air with several reso-
nant biosensors. An appropriate cell fixation procedure was applied
prior to microwave measurements to maintain their intracellular
content close to live state while measuring the cells outside of their
aqueous and ionic medium.
In the following sections, we will discuss the biosensor opera-
tion principle, design and fabrication. Material and methods used
for this study will also be detailed. Finally the data collected though
experiments performed on the five selected colorectal cancer cell
lines will be presented and discussed.
2. Label-free biological cell sensing sing microwaves
2.1. Microwave detection principle and sensor design
The microwave spectrum has long been considered as a promis-
ing frequency domain for cellular analysis. Indeed, water-based
samples, such as biological cells, are known to exhibit strong dielec-
tric property dispersion at such frequencies [17]. Hence, according
to their tissue of origin and/or pathological state, it has already
been shown that cells seem to display significant differences in
their responses to microwave stimuli [14,18]. In previous works
[23–25], the authors have developed microwave biosensors able to
achieve dielectric spectroscopy analysis at the cellular scale. Once
carefully calibrated and properly used, such sensors allow measur-
ing the average intracellular dielectric permittivity dispersion over
frequency, and thus establishing associated electromagnetic (EM)
signatures for selected cells. Fully label-free, this method allows an
accurate dielectric intracellular analysis performed at the unicellu-
lar scale.
To design these sensors, a meandered inductor and two inter-
digitated comb (IDC) capacitors are set in parallel and implemented
in a microwave coplanar waveguide as illustrated in Fig. 1 pho-
tographs. From an electromagnetic point of view, such device
works as a stop-band LC resonator for which the transmitted signal
through the device is strongly attenuated when the sensor res-
onates. Therefore, the LC resonator S21 parameter, which relies on
sensor frequency response, shows a minimum value at this specific
resonance frequency. Indeed, S21 is the ratio between amounts of
RF power transmitted though the sensor and amounts of power
applied at the sensor input.
The main reason for using resonant microwave sensors for this
study is their ability to achieve an accurate microwave dielectric
spectroscopy analysis even if a very low biological samples num-
ber are involved. Indeed as illustrated in Fig. 2 example where only
three cells load the sensor, such design allows benefiting from high
detection sensitivity insofar as any dielectric perturbation on the
comb capacitors results in a sensor resonance change that can be
easily observed and measured: the resonance frequency is shifted
to a lower frequency and a slight microwave signal attenuation
change occurs at the resonance. Hence, to assess the own cell intra-
cellular medium permittivity with a relatively good accuracy at
this scale, we monitor sensor resonance frequency changes, once
cells are deposited or cultured on sensing IDC capacitances. In fact,
when sensor capacitors are loaded with few dielectric particles, the
resulting resonant frequency shift depends on three main param-
eters: the number of particles involved, their individual size and
associated volume, and the average relative dielectric permittivity
value of their content considered at the sensor resonance frequency
[23]. Now, taking into account these three parameters, the reso-
nant biosensors can be calibrated to become sensitive to the nature
and pathological state of analyzed cells. Using an appropriated
modeling, the dielectric properties of individual cells can be then
estimated at the resonance frequency of each sensor used. With
such approach, an EM signature over frequency can be established
for investigated cells. As it is shown in this paper, this signature
seems to be representative of the original type and pathological
state of cells.
 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416 407

Fig. 1. Main characteristics of few passive resonant sensors used in this study, including for each of them: a microscope view illustrating geometry design changes between
sensors, the unloaded LC resonator frequency response (before cell deposition), and the associated equivalent electrical model with computed circuit element values.

Fig. 2. Resonance frequency shift measured on S1 type passive sensor once loaded with 3 DLD-1 cells; also including a bottom microscope (upper panel) and a scanning
electronic microscope (SEM) (lower panel) views of the interdigitated capacitor onto which cells have been deposited, fixed and dried.

To collect the experimental data presented in this work, two different passive sensor designs of which four are shown in Fig. 1.
biosensor design families were employed. The first one is based This sensor type, allows characterizing the intracellular cell dielec-
on a passive LC resonator design that presents a fixed reso- tric permittivity only at a single frequency. As discuss previously,
nance frequency. For this study, we have worked with around ten this frequency actually corresponds to the sensor resonance. To
408 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416

Table 1 500 MHz continuous frequency tuning. In fact for present sensors,
Measured resonance frequency vs varactor bias voltage for active sensors.
this tuning range is intrinsically limited by the moderate capac-
Bias (V) itance tuning ratio (∼2) achievable with MA46H146 varactors.
However, other alternative tuning components can be considered
0 0.5 1 1.5 2 2.5 3 4 5
for this application. As an example, it would be interesting to use
Sensor SA
digitally tunable capacitors with fine tuning step and high Q perfor-
f0 (MHz) 4954 5107 5212 5284 5344 5398 5437 5506 5557
Sensor SB mance in order to enlarge the analysis sensor bandwidth. Such high
f0 (MHz) 5668 5782 5872 5941 5995 6043 6088 6121 6151 performance capacitor banks are indeed now available on CMOS
Silicon-On-Sapphire [26] or RF-MEMS technologies [27], and their
integration on sensors would result in a compromise between tun-
obtain wideband EM signatures on a cell line, several cells of this able component size and connectivity constraints, and the sensor
line have to be analyzed with different sensors working at differ- frequency tuning, sensitivity and detection capabilities.
ent frequencies. In this case, we used sensors presenting different Another solution is to combine several sensors with different
meander inductor geometries (i.e. different inductor length) that inductor geometries. Indeed, with a proper design, the covered
allow us spreading their resonance frequency over the 5–14 GHz analysis spectrum can be easily enlarged using sensors presenting
band for the present study. adjacent frequency bandwidths as in the example of the SA and
Actually, in order to limit the number of sensors required to SB sensors shown in Fig. 3. Hence, the number of sensors required
establish a cell line EM signature, frequency tunable resonators to establish a representative and wide band EM signature can be
were developed. This second sensor generation integrates an active strongly reduced.
element allowing adjustment of the LC resonator’s capacitor value,
making the sensor resonance frequency tunable (Fig. 3). Hence, 2.2. From measured sensor resonance frequency shift to cell
the sensor working frequency can be continuously adjusted on a electromagnetic signature plot
predefined band. It means that, for each frequency of this band,
it becomes possible to assess the expected cell dielectric proper- In this section, the methodology used to establish the cell EM sig-
ties measured this time starting from the same biological samples. nature is detailed and discussed. Actually, to obtain a representative
In the present case, the active component is a surface-mounted signature, we have seen that several sensors are needed in order to
semiconductor diode varactor (MA46H146 from MA-COM). As ensure cell dielectric property measurements on a sufficiently wide
illustrated in Fig. 3, this tunable capacitor is set in parallel with frequency band. Hence during this measurement campaign, each
the LC resonator. of these sensors is loaded with cells derived from the same line. At
The capacitance value can be changed by applying an appropri- the end, the dielectric permittivity of the intracellular content of
ated DC voltage to the diode through an integrated biasing network selected cell is computed using to the resonance frequency change
(biasing resistor and DC block capacitance). More details on the sen- detected and recorded on each of these different sensors.
sor design and operation can be found in [24]. As detailed in Table 1, During microwave measurements, there is a quasi-uniform
the frequency tunable sensors used for this study exhibit at least electromagnetic field between the used sensing electrodes. Indeed,

Fig. 3. Microscope view of the three tunable resonance frequency sensors used in this work with their tunable unloaded LC resonator frequency responses (before cell
deposition) and their associated equivalent electrical models with computed circuit element values.
 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416
Table 2
Main characteristics of investigated cell lines.
Cell Line WiDr SW480
Stage (Duke’s classification) II II
Diameter (mean value) 12.7 ␮m 13.1 ␮m
Diameter (median value) 12.4 ␮m 12.9 ␮m
Cell adhesion on glass +++ ++
Cell morphology
as it will be detailed later on in this paper, this is made possible
thanks to the polymer coating which covers the sensor surface. As
one can see in Fig. 5, in this polymer a window is specifically opened
above each sensing capacitor to form a small micro-chamber. Actu-
ally, regarding the design of this chamber, all parts of IDC electrodes
which present a non-uniform electromagnetic field distribution
are hidden under the polymer. As a result, whatever the cell loca-
tion between and along the comb capacitance fingers, its effect
and influence on sensor will be absolutely the same. Moreover,
one can observe in scanning electronic microscope image shown
in Fig. 2 that once cells have adhered on the substrate right at
the micro-chamber bottom; they generally entirely fill the air gap
width between IDC electrodes. This is mainly due to the fact that
the sensor drying favors a cell drop down and flattening in the
bottom of capacitor interdigitated gaps. Hence, once cells are load-
ing a sensor, the applied microwave electromagnetic field should
interact with the whole cell cytoplasm, including proteins, nucleic
acids, electrolytes and water content. To fulfill this objective, the
current sensor configuration demands to work outside of an aque-
ous environment. Therefore, we chose to perform measurements
on fixed cells, which are more likely to handle such environ-
mental constraints without alteration. For our experiments, we
selected a formaldehyde fixation protocol that is considered as a
gold standard by biologists to study cell morphology, organelles,
proteins (antigens) and nucleic acids, even if not the best suited
for some particular applications [29]. Aldehydes are commonly
employed in biology to prepare tissues and cells for manipulation
outside of a survival medium. They act by crosslinking proteins
and thus “freezing” a tissue or cell in its native state. Here, we are
working in a single-cell rather than a whole tissue context. How-
ever, contrary to whole tissues, the effects of fixation procedures
at a single-cell level, including formaldehyde, have been relatively
understudied. The protocol that we have chosen ensures that water
content does not leak out of the cell through an altered membrane,
at least for a timeframe that we will discuss later.
Finally, the induced perturbation occurring on the sensor res-
onance is the result several effects: the amount of water present
in cell cytoplasm, its ionic and protein concentrations, the size and
own electrical properties of the nucleus and other organelles, etc.
The experimental data we can collect with our biosensors do not
allow us discriminating one of these influences from the others.
That is why we will consider in following calculus only average
dielectric properties for the whole cell. Indeed, we model cells as
homogeneous dielectric particles, defined with their own dielec-
tric permittivity; this dielectric permittivity is supposed to follow
a dispersive frequency dependence as it is commonly observed in
any biological samples holding water [17,30]. Moreover, this model
does not consider the thin cell membrane, since it can be seen as
transparent in the frequency domain of interest [12].
We also assume that the large majority of cells, coming from the
same line, possess identical dielectric permittivity characteristic.
409
SW620 DLD-1 Colo 205
III III IV
11.4 ␮m 13.6 ␮m 12.8 ␮m
11.2 ␮m 13.5 ␮m 12.7 ␮m
++ ++ +
Insofar as they come from the same origin and during their culture
they were not subject to any specific treatment, they should keep
the same pathological state. We also consider an identical intracel-
lular volume for each cell coming from the same line. This volume
is computed from size measurements done on a representative
cell population (20,000 up to 50,000 cells) using a Coulter Counter
apparatus. One should notice that there is inevitably a size distri-
bution on such living cell population; actually standard deviation
generally ranges between 1 and 2 ␮m on cell diameter measure-
ments. Nevertheless, as we can notice in data reported in Table 2,
the difference between the mean cell diameter and its median value
is very small (<2.5%) for the investigated cell lines. Its means, that
the cell size dispersion actually follows very narrow Gaussian dis-
tribution and a large cell number may have very close intracellular
volume. Indeed, immortalized cell lines are well known to show
homogeneous morphological characteristics unlike cells coming
from primary cultures. In the end, it seems that the induced error
on the cell dielectric permittivity value might remain acceptable if
we consider the same intracellular volume for one cell to another
as long as they come from the same cell line.
Now, to establish representative EM signatures of selected cell
lines, we focused on computations of the real part of the cell relative
permittivity, which gives relevant information on the intracellular
content specificities as shown in the next section. The imaginary
part of the cell permittivity, which relies on the intracellular dielec-
tric loss and ionic conductivity, would be also very interesting to
know. However, our sensor detection capabilities do not currently
allow us to measure its effect on the sensor frequency response
with a satisfactory and reproducible accuracy.
In our previous works, 3D fullwave EM finite element model-
ing of both unloaded and cell loaded sensor were used to extract
the real part of the cell relative permittivity from measurements
[22,23]. Such EM computation allowed accurately fitting the sen-
sor measured frequency response with the cost of a quite long
simulation time. However, for this study, an analytical model was
developed to compute the real part of the intracellular permit-
tivity in real-time during experiments. In this model, the cell is
assimilated to a uniform dielectric block located between two inter-
digitated capacitor fingers. As illustrated in Fig. 4, to an electrical
point of view, this dielectric block acts in fact as an additional capac-
itor CCell that loads the IDC sensing capacitor and increases its initial
value. And, if several cells are involved, their capacitive influence
will be cumulative. As a consequence, the LC resonator sees its reso-
nance shifting from frequencies f0 to f1 ; both given by the following
equations:
1
f0 =  (1)
2 LCU
with CU = C0 + C0 or CU = C0 + CVar considering respectively passive
or active sensors where C0 is initial capacitance of IDC capacitor
410 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416

Fig. 4. Modeling of cell capacitive influence on sensor IDC capacitor; used sensor comb fingers are 4–5 ␮m thick and spaced 13 ␮m (=WIDC ) from each other.

before cell loading (Fig. 1) and Cvar is the varactor capacitance value
(Fig. 3).
1 mittivity of cell can be computed using the following equation:
f1 =  (2)
2 LCL
 Cell
with CL = CU + Ccell where CU and CL respectively represent the
overall sensor capacitor without and with cells loaded on and L
models the resonator inductive behavior.
Hence, knowing the unloaded and loaded sensor resonant fre-
quencies (f0 and f1 ) and the unloaded initial sensor capacitor CU ,
the resulting cell capacitor CCell can be computed using Eq. (3) that
results from combining (1) and (2).
 CU (f0 − f1 )(f0 + f1 )
CCell = Ncell · CCell = 2
(3)
f1
where NCell represents the number of cells actually loaded on the
sensor.
In practice, the CU capacitance value is precisely extracted
thanks to dedicated test structures based on LC resonator design
for which the meander inductor was removed.
Now, assuming that the cell volume VCell remains constant dur-
ing measurement and assuming that all cells entirely fill the air gap
width (WIDC ) between two IDC fingers, the real part of the relative
permittivity is then given by:
where ε0 is permittivity of free space.
Finally, combining (3) and (4), the real part of the relative per-
WIDC 2 (f0 − f1 )(f0 + f1 )
εr = CU · (5)
f12 · ε0 · VCell · NCell
Regarding this formula, one can notice that the accurate mea-
surement of sensor resonant frequencies f0 and f1 is an important
issue. In order to limit resulting errors on the cell permittivity
computation, a polynomial curves fitting technique is applied on
sensor microwave measurement data. Combined with the 1 MHz
frequency resolution used during RF measurement, this method
allows us determining the sensor resonance frequency with a
satisfactory accuracy (i.e. error less than 5 MHz). During sensor
measurements, the exact number of cells loaded on sensing capaci-
tors was systematically controlled under microscope. As previously
discussed, their exact location inside the gap is not an important
issue and does not change the sensor resonance shift. Consequently,
the main source of uncertainty comes from the accuracy of the
intracellular volume estimation as it has been previously discussed.
3. Materials and methods
3.1. Cells preparation

CCell · WIDC 2 Experiments presented in this paper were performed with five
εr Cell = (4)
ε0 · VCell · NCell distinct cell lines (WiDr, SW480, SW620, DLD-1 and Colo 205)
 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416
bought from the ATCC (American Type Culture Collection). All of
them are human colorectal cancer cell lines derived from low
to high grade tumors (colorectal adenocarcinoma) [31]. They are
derived from colon epithelium tissue and all originated from an
adult patient. The SW480 and SW620 lines were especially derived
from the same patient; SW480 was isolated from the primary ade-
nocarcinoma arising in the colon, whereas SW620 was isolated
from a lymph node when the cancer recurred with wide spread
metastasis few years after SW480 origin cells was collected. SW480
is considered a grade II while SW620 is a grade III. These five lines
are commonly used as biological models since they are considered
to be representative of increasing aggressiveness cells involved in
increasing malignant stages of colorectal tumor [31–34]. Table 2
sums up the main characteristics of these cell lines, including the
average size used to estimate their intracellular volume during our
computation. One can also see in Table 2 that adhesion properties
of these cells decrease with increasing aggressiveness stage. This
is why two different techniques were developed to drop off cells
inside the biosensors micro-chambers according to their adhesion
capability on glass.
Regarding cells with good adherence properties, the principle
was to deposit and grow cells directly on the biosensor surface.
With this purpose, biochips were placed into six-well plates and
submerged in the appropriate culture medium. A well-controlled
number of cells were then seeded into the well to ensure up to five
or six cells adhered on the sensor capacitive slots located inside
the micro-chambers. Two days of culture were generally required
at 37 ◦ C in a humidified 5% CO2 – 95% air incubator. As it will be
discussed later, since cells were characterized outside their cul-
ture medium, they were beforehand directly fixed on the biosensor
using a formaldehyde solution (FA 4% in water). This fixation kills
cells but allows maintaining their membrane integrity and their
intracellular content as close to living condition [28] [29]. Sensors
were then rinsed and immerged in deionized (DI) water.
Concerning cell lines with poor adherence properties, they were
quite difficult to directly grow on the sensor surface as a significant
number were lost during the final washing and drying steps. An
alternative protocol was thus developed. These cells were conven-
tionally grown in cell culture flask (37 ◦ C in a humidified 5% CO2 –
95% air incubator). After two days of culture, cells were collected,
fixed in 4% FA, rinsed three times in DI water and resuspended in
DI water. Finally, a small droplet (∼50–100 nl) of this cell suspen-
sion was deposited inside the biosensor micro-chambers thanks to
a glass capillary connected to a syringe pump. During the droplet
evaporation, cells dropped down and placed themselves inside the
gaps formed by capacitor electrodes thanks to strong induced cap-
illary forces occurring while the droplet was drying.
In the end, the number of cells loaded on the sensor also impacts
on the measurement accuracy. Indeed, we had to be sure that
loaded cells are well inserted inside the interdigitated gaps to
ensure an optimal interaction with the microwave electric field and
then disturb the sensor resonance. In cases for which a cell covers
or overlaps a sensing electrode as well as when it does not effi-
ciently fill the gap, the resulting resonance frequency shift is not
as large as it can be if this cell is properly placed between two IDC
electrodes. Consequently cell dielectric properties cannot be deter-
mined with a satisfactory accuracy in any of these configurations.
Actually, sensors are carefully controlled under microscope. Every
time a sensor encountered such problems or if we had any doubt
about a cell position on sensing capacitors, the measured data was
not considered to establish the cell line EM signature. That is why,
for this study, we were looking for limiting the number of cells
loaded on the sensor to few ones (typically less than ten). In this
case, the risk to form cell aggregates was very limited, microscope
controls became easier and more reliable, and we expected in this
way to reduce measurement errors considering as an example the
411
Fig. 5. SEM view of passive resonant sensor covered with a 40 ␮m thick SU8 poly-
mer; the sensor is tilted with a 45◦ angle. The inductance is here hidden due to SU8
thickness (most of electrons are absorbed in the polymer). This picture shows only
surfaces on which cell can adhere.
wrong number of cells which really affected the sensor frequency
response.
So, to regulate the number of cell involved during sensor loading,
the initial cell concentration and culture time were optimized when
cells were cultured directly on sensor. Whereas, when droplet
deposition technique was used, we adjusted the concentration of
cells suspended in DI water to restrain the average number of float-
ing cells in each droplet.
3.2. Biosensor fabrication
Biosensors used in this work were fabricated on fused silica
substrates using biocompatible materials and a standard micro-
electronic process. This process allows fabricating 4–5 ␮m-thick
electroplated gold electrodes that are patterned with UV lithog-
raphy to make the LC resonator designs. As illustrated in Fig. 5, a
thick SU8 polymer (40 ␮m) is then used to cover most of these gold
electrodes; except on the sensor capacitive detection areas and the
coplanar waveguide access that are used to inject the microwave
signal in the sensor thanks to RF probes. In practice, this protecting
layer is thick enough to avoid RF signal interaction with any cell
accidentally located outside the sensor detection areas. Indeed, the
SU8 polymer forms two dedicated micro-chambers on the sensor
surface, in which cells were concentrated during their culture on
sensors or directly deposited with the droplet technique.
These micro-chambers are patterned on the LC resonator
sensing capacitor, where most of the microwave electric field is
concentrated at the resonance frequency [22]. An interesting point
comes from the fact that during the cell culture, cells generally pre-
fer to adhere on the fused silica substrate rather than on SU8. Hence,
we frequently observed cells located on the SU8 slowly moving to
drop down into the opened windows in this polymer and adhere
between RF sensing electrodes.
These micro-chambers were also useful when cells are
deposited on sensors using the droplet technique. Indeed, they
allowed restricting the droplet spreading only on the IDC sensing
area. Actually, droplets of cell suspension were injected inside the
200 ␮m × 200 ␮m micro-chambers thanks to a glass capillary with
a 120 ␮m outside diameter until micro-chambers volume was com-
pletely filled. Hence, we succeeded to obtain good reproducibility
of the time required to fully evaporate the DI water droplet and
make deposited cells adhere on sensor.
412 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416

3.3. RF electromagnetic signatures measurement methodology

Before culture or cell deposition in micro-chambers, biosensors

needed to be first of all calibrated. For that, the four S-parameters
(i.e. S11 , S21 , S12 and S22 which represent the whole frequency trans-
fer function of the sensor) were measured for each of them and
recorded as a reference (unloaded S-parameters). These measure-
ments required standard microwave lab equipment. In the present
case, we used a probe station (Süss microtec) equipped with GSG
(Ground Signal Ground) RF probes (Cascade ACP type) and a net-
work analyzer (R&S ZVA-24) calibrated using the TOSM method (i.e.
using Through, Open, Short, and Match standards). In this study,
we mainly worked in the 5–14 GHz frequency band with mea-
surements done every 1 MHz step. In order to measure the sensor
the S-parameters, a microwave signal with a −10 dBm (0.1 mW)
power magnitude was applied at the sensor input. This has ensured Fig. 6. Examples of measured resonance frequency shifts at 22 ◦ C after SW620 cells
that cells under characterization did not handle possible thermal were grown on passive sensor chip.
effects due to microwave [35], which might disturb the biosensor
response. Hence, at the sensor resonance where the electric field SW620 cells, the measured frequency shifts were about 75 MHz for
was maximum between sensing electrodes, the resulting electric the first one (i.e. a fractional shift of 1.25%), 40 MHz for the sec-
field applied on cells ranged between 10 and 20 kV/m. ond one (i.e. a fractional shift of 0.55%), 89 MHz for the third one
Conventional biological media used for cell culture or handling (i.e. a fractional shift of 0.94%) and 112 MHz (i.e. a fractional shift of
are aqueous saline solutions. It is well known that such media 0.90%) for the fourth one. Considering the number of cells loaded
present a strong absorption in the gigahertz domain [36–38]. onto each sensor and their average volume, the analytical model
Consequently if such medium is still present inside the analy- (Eq. (5)) allowed us to compute the resulting intracellular average
sis micro-chamber, a strong degradation of the biosensor quality permittivity for each investigated frequency.
factor occurs when it resonates. In these conditions, the sensor We repeated these experiments several times with different
detection capabilities and sensitivity may completely disappear in sensors; the whole data collected are plotted in Fig. 7. One can
worst cases. This is the reason why, all microwave characteriza- see that the computed relative dielectric permittivity of these cells
tions were performed on cells removed from their support media. clearly decreased when the frequency rose. This tendency is actu-
Indeed, once loaded with cells, all sensors were dried in ambient air ally intrinsically linked to the intracellular water content and its
before running microwave measurements. In practice, we waited dispersion behavior over frequency [39]. A Cole–Cole equation,
50–90 s once the entire support medium was fully evaporated from such as (6) if we are interested only in the real part of the dielectric
micro-chambers to perform measurements. For each measurement permittivity, can be used to model this frequency dependence with
campaign, this drying time was evaluated using reference sensors a good accuracy [40] and might allow extracting average tendencies
for which small volume of DI water was deposited inside micro- or statistical data from experiments as it is done in [18].
chambers several times. We then measured the time required to
recover the initial resonance frequency of reference sensors. εr (1 + (ω)1−˛ sin(˛/2))
εr Cell (ω) = + εr·∞ (6)
Depending on the tested cell lines, the microwave measure- 1 + 2(ω)1−˛ sin(˛/2) + (ω)2−˛
ments in ambient air of sensors loaded with cells can be done until
7–15 min after drying. Indeed, during this time the measured sen- where ω is the angular frequency (i.e. ω = —
— 2f) and εr∞ , εr ,  and
sor S21 parameters were very stable with no noticeable drift of ˛ are variable parameters chosen to fit the experimental data.
resonance frequency. Beyond this period, we suddenly observed a As plotted in Fig. 7, such model seems to show a good agreement
change on the sensor resonance frequency which recovered almost with our experimental data (considering here εr∞ = 3, εr = 97,
the initial frequency before be loaded with cells. We assumed that  = 27 ps and ˛ = 0.005) and might be used to determine an aver-
this phenomenon might occur when the cell membrane finally age electromagnetic signature representative of the SW620 cell line
broke letting then escape and evaporate the whole cytoplasm liq- properties.
uid outside the cell. Actually, the dehydration of investigated cells
had been delayed thanks to the applied FA fixation letting us greatly
enough time to properly characterize cells in air condition. Indeed,
less than 30 s were required to record the sensor S-parameters.
Hence, in the case of experiments for which cells were deposited
with the droplet technique, S-parameters were measured no later
than 2 min after suspended cell droplets had been deposited inside
micro-chambers. Whereas for experiments done with cells cul-
tured directly on sensor, all the sensors fabricated on the same
chip were measured during the 5 min following the end of chip
drying. It is important to notice that, without fixation, it was no
possible, whereas cells were taken out a proper osmotic aqueous
medium, to efficiently maintain the intracellular content integrity
during sensor microwave measurement.
As an example, the frequency responses observed on four differ-
ent passive biosensors before and after being loaded with SW620
line cells are shown in Fig. 6. Without any cell, the biosensor
resonant frequencies were 5.993 GHz, 7.263 GHz, 9.481 GHz and Fig. 7. Collected data on SW620 cells: real part of their relative permittivity vs
12.448 GHz respectively. Once loaded with one, two or even three frequency.
 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416 413

Table 3 -12
Computed relative permittivity of SW620 cell line from collected experimental data
obtained with on chip culture and droplet deposit techniques.

On chip culture technique

f1 (MHz) 5993 7263 7709 7786 9446 -15

εr 52 ± 5 41 ± 4 41 ± 4 43 ± 4 34 ± 3

S21 (dB)
Loaded sensor (5 SW620 cells)
94 MHz
Droplet deposit technique Unloaded sensor
f1 (MHz) 6022 7228 7700 7741 9300 -18 0.39 dB Loaded sensor (5 WiDr cells)
εr 54 ± 5 40 ± 4 43 ± 4 39 ± 4 29 ± 3

Unloaded sensor
0.32 dB
One should notice that the experiments achieved on the SW620 64 MHz
cell line and summarized in Fig. 7 were led using the two deposition -21
8 9 10 11 12
techniques: on-chip cell culture and floating cell droplet deposi-
Frequency (GHz)
tion. We can see in Table 3 that whatever the technique, collected
experimental data both were in good agreement: the extracted rel- Fig. 8. Measured resonance frequency shifts at 22 ◦ C on SW620 and Colo 205 cells
ative permittivity was in the same range for the two approaches. were loaded onto the sensors.
These results gave us confidence on the insignificant influence
of the deposition method on EM signatures extracted from tumor (grade II). For these characterizations, only passive sensors
measurements. have been used, with the on-chip cell growth technique. Fig. 8
focuses on two measurements done using two sensors with compa-
4. Results and discussion rable unloaded resonance frequencies, and each sensor loaded with
the same number of cells. Indeed, the first one was loaded with five
A tumor stage is defined with respect to anomalies at the tissue WiDr cells, whereas the second one was loaded with five SW620
scale and invasion of suspect cells inside healthy tissue. Among the cells. As detailed in Table 2, these two cell lines present similar
large number of malignant cells involved in tumor tissues, some intracellular volumes and measurement conditions were the same
of them show stronger aggressiveness than others. They may have for both sensor S-parameter measurements (i.e. room tempera-
the ability to proliferate very quickly, regenerate a whole tumor, ture, VNA calibration, and equipment). Consequently, considering
and even produce metastases in remote tissues. The aggressive- Eq. (5), the observed frequency shift difference between these two
ness of cells making up a tumor may be correlated to the tumor sensors mainly relies on intrinsic dielectric properties differences
stage. Hence, high-grade cancer generally involves a large num- between the two cell lines.
ber of highly aggressive cells [31]. The main objective of this work As for SW620 line, the estimated dielectric permittivity for WiDr
was to determine if electromagnetic signatures of malignant cells cell followed a dispersive behavior as frequency increases (Fig. 9).
derived from low to high grade tumors could present some notice- As discussed in the previous section, this phenomenon stems from
able differences and if these signatures could be correlated to a cell intracellular content dielectric properties, largely dominated by
aggressiveness level. For demonstration purpose, five cancer cell
lines (WiDr, SW480, SW620, DLD-1 and Colo 205) derived from
human colorectal cancer at different stages have been selected.
Fig. 7 illustrates the results obtained from experiments led on
SW620 line (derived from grade III tumor). In this case, data was
recorded during four measurement campaigns following strictly
identical cell preparation conditions. Data was obtained using fif-
teen passive sensors (data labeled with squares) and four tunable
frequency sensors (data labeled with circles, triangles or dia-
monds). To make reading easier, error bars were not plotted for
these last ones; nevertheless, they were within the same range
as passive sensors. More details related to active sensors results
are reported in Table 4 regarding the two sensors previously
introduced in Fig. 3 and Table 1. In the end, data extracted using pas-
sive or tunable frequency sensors were in good agreement, while
tunable frequency sensors allowed collecting a much larger amount
of data from a single experiment.
Similar measurement campaigns have been conducted on a Fig. 9. Real part of relative permittivity extracted from measurements for WiDr and
WiDr cell line derived from a lower aggressiveness stage colon SW620 cells.

Table 4
Real part of SW620 cell dielectric permittivity (εr ), computed from data collected using two tunable frequency sensors.

Bias (V)

0 0.5 1 1.5 2 2.5 3 4 5

Sensor SA
f1 (MHz) 4924 5074 5179 5248 5308 5362 5401 5470 5521
εr 62 ± 7 62 ± 7 59 ± 6 61 ± 6 59 ± 6 58 ± 6 56 ± 6 54 ± 5 53 ± 5
Sensor SB
f1 (MHz) 5647 5761 5851 5920 5974 6022 6064 6130
εr 57 ± 6 54 ± 5 51 ± 5 50 ± 5 55 ± 6 54 ± 6 53 ± 6 52 ± 5
414 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416
Fig. 10. (a) Real part of relative permittivity extracted from measurements and
Cole–Cole modeling for WiDr, DLD-1, SW620 and Colo 205 cells. (b) Representa-
tion of the possible correlation occurring between malignant cell EM signatures and
their potential aggressiveness degree.
the amount of water in the cell cytoplasm. An interesting learn-
ing comes from a failed characterization campaign achieved on
WiDr cells, which is also illustrated in Fig. 9. During this campaign,
some problems occurred during the cell fixation step; cells were
not properly fixed, and they ended up strongly degraded once the
sensors had dried. Indeed, for the different sensors used, we never
succeeded in observing stable resonance frequency. Instead, sen-
sors showed unstable frequency shift that rapidly changed until
almost disappearing as if intracellular content evaporated during
the few seconds after complete sensor drying. Sensor measure-
ments have been done once S-parameters were stabilized. The
extracted resulting dielectric permittivity was very low (i.e. less
than 10) and did not show any frequency dispersion. These results
suggest that measured permittivity differences between cell lines
are due to noticeable changes within the intracellular medium.
Regarding the error margins that were considered, uncertain-
ties on the cell volume and positioning in the sensing IDC slots
as well as on accurate resonance frequency shift measurements
were taken into account. Fig. 9 shows that the computed WiDr
line relative dielectric permittivity was always less than SW620
between 5 and 14 GHz. Consequently, as we initially assumed, it
seems that some EM signature differences exist between these cell
lines. Therefore, a relationship between intracellular content and
tumor aggressiveness stage might be established. This hypothesis
appears to be confirmed by data collected on the SW480 line that
originates from the same patient as SW620 cells, but from a lower
grade tissue (grade II). As reported in Fig. 10a, the EM signature
Table 5
Used Cole–Cole equation parameters to model measured cell permittivity dispersion
as function as frequency.
Parameters SW480 WiDr SW620 DLD-1 Colo 205
εr∞ 3 3 3 3 3
ε 87 87 97 97 97
 34 ps 30 ps 27 ps 23 ps 17 ps
˛ 0.005 0.005 0.005 0.005 0.05
established for SW480 cells also clearly differs from SW620. One
can notice that the related dielectric permittivity of SW480 cells is
much lower than SW620, as in the WiDr case. This allows us assum-
ing that the tumor grade of the tissue of origin may influence the
dielectric permittivity detected with our sensors.
This hypothesis was further supported by results obtained on
two other cell lines derived from higher grade tumors: DLD-1
(grade III) and Colo 205 (grade IV). Indeed, as presented in Fig. 10a,
computed permittivity frequency dispersion trends clearly show
differences between the DLD-1 and Colo 205 lines; this is also
true for SW480, WiDr and SW620. These additional signatures
confirmed that malignant cells derived from high tumor grade
exhibited the highest dielectric permittivity values, while cells
derived from low grade models displayed much lower permittiv-
ity values. Table 5 summarizes the modeling parameters used to
fit experimental data with Eq. (6) derived from a Cole–Cole model.
These curves are plotted in Fig. 10a for each investigated cell line.
Form this data, it is difficult to draw a general conclusion. Still, one
can notice a trend regarding the time constant parameter  used to
model the frequency dependence of the different lines investigated.
Indeed, in the 5–14 GHz band, the frequency dispersion of dielec-
tric properties (i.e. the variation of measured permittivity values vs
frequency) appears to be lower for cells derived from higher grade
tissue than for cells from lower grade tissue.
However, the differences in EM signature observed between
cells derived from tissue staged at the same grade are currently
not fully explained. It may rely on intracellular features proper
to each considered biological model. Especially, water content
variation related to the overall cell volume or even changes in
organelles/nucleus (size, number, physical properties, etc.) may
explain the differences in dielectric characteristics we observed
for the investigated cell lines. Additional experiments involving
nucleus vs cytoplasm volume ratio measurements or careful anal-
ysis of cytoplasmic ions, macromolecules and proteins might help
gaining more insight into our results.
Nevertheless, we believe that the preliminary results presented
in this paper are very promising; as they tend to show that the
intracellular EM signature may provide information about the
aggressiveness potential of cells known to be malignant. Indeed, as
illustrated in Fig. 10b, it may be possible to define some dielectric
permittivity ranges that may be associated a cell aggressiveness
degree. In fact, such aggressiveness degree may be correlated to
a more or less high probability that tumor holds some cells with
highly invasive capabilities. It is clear that larger investigations in
the frame of a clinical study supported with statistical analysis will
be required to properly define these ranges and evaluate the rele-
vance of this idea. However, if these results are confirmed, it may
be possible to use the analysis of cell dielectric property as a novel
prognosis indicator for the course of the disease that might be com-
plementary to conventional anatomo-pathologic tumor diagnosis.
Our current circuit designs can be greatly improved for prac-
tical clinical use. We also believe that microfluidic technologies
can be helpful for single cell analysis in a flow condition. To our
point of view, it should be a very good approach for characteriz-
ing a large number of cells required to be representative of the cell
population heterogeneity that makes up a tumor. Challenges are
 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416
that sensors have to ensure both accurate flowing cell detection
in dispersive liquid media and analysis of their own intracellular
dielectric properties.
5. Conclusion
This paper demonstrates the capability of microwave resonant
sensors to distinguish some differences in colorectal tumor cells
from cell scale permittivity measurements. More specifically, the
presented work is a proof-of-concept study based on experiments
achieved on immortalized cancerous cell lines. It demonstrates
the potential of intracellular dielectric permittivity analysis at
microwave frequencies to evaluate the degree of aggressiveness
on malignant cells. The results presented here show a potential
relationship between the tumor grade and the resulting EM signa-
ture of these cells. We are currently working on extending these
preliminary results with investigations on primary culture cells or
cells coming from dissociated fresh cancer patient tissues in order
to prepare future clinical trials.
The high detection sensitivity achieved on such microwave res-
onant biosensors allows working at the single cell level. As a result,
such label-free biosensing technology is really promising to target
uncommon or rare cells and to provide an early prognosis with the
objective to assess and prevent tumor spreading and metastasis
development. In this view, such biosensors could reveal a new and
powerful tool to complete conventional medical analysis process,
particularly for early detection of cancer.
Acknowledgments
This work was supported by the French Limousin Regional
Council (Région Limousin) the European Commission with FEDER
(Fonds Européen de Développement Régional). The authors are
grateful to D. Passerieux for his help and expertise during RF mea-
surements and F. Sidor and C. Guines for their assistance during
biosensor fabrication.
References
[1] C.J. Choi, et al., Comparison of label-free biosensing in microplate, microfluidic,
and spot-based affinity capture assays, Anal. Biochem. 405 (1) (2010) 1–10.
[2] J.S. Daniels, N. Pourmand, Label-free impedance biosensor: opportunities and
challenges, Electroanalysis 19 (12) (2007) 1239–1257.
[3] A. Treizebre, et al., Terahertz spiral planar Goubau line rejector for biological
characterization, Prog. Electromagnet. Res. M 14 (2010) 163–176.
[4] H.J. Mulhall, et al., Cancer, pre-cancer and normal oral cells distinguished by
dielectrophoresis, Anal. Bioanal. Chem. 401 (8) (2011) 2455–2463.
[5] Y.I. Kim, et al., Biosensors for label free detection based on RF and MEMS tech-
nology, Sens. Actuators B: Chem. 119 (2) (2006) 592–599.
[6] L.A. Flanagan, et al., Unique dielectric properties distinguish stem cells and their
differentiated progeny, Stem Cells 26 (3) (2008) 656–665.
[7] C.E. Sims, N.L. Allbritton, Analysis of single mammalian cells on-chip, Lab Chip
7 (4) (2007) 423–440.
[8] S. Nagrath, et al., Isolation of rare circulating tumor cells in cancer patients by
microchip technology, Nature 450 (2007) 1235–1239.
[9] A. Rasooly, J. Jacobson, Development of biosensors for cancer clinical testing,
Biosens. Bioelectron. 21 (10) (2006) 1851–1858.
[10] M. Ponz de Leon, et al., Clinical and pathologic prognostic indicators in colorec-
tal cancer, Cancer 69 (3) (2006) 626–635.
[11] A. Jemal, et al., Cancer statistics, CA: Cancer J. Clin. 59 (4) (2009) 225–249.
[12] K. Asami, T. Yonezawa, Dielectric behavior of wild-type yeast and vacuole-
deficient mutant over a frequency range of 10 kHz to 10 GHz, Biophys. J. 71
(1996) 2192–2200.
[13] K.R. Foster, J.L. Schepps, Dielectric properties of tumor and normal tissues at
radio through microwave frequencies, J. Microw. Power 16 (June (2)) (1981)
107–119.
[14] D.-S. Yoo, The dielectric properties of cancerous tissues in a nude mouse
xenograft model, Bioelectromagnetics 25 (October (7)) (2004) 492–497.
[15] H. Scharfetter, Structural Modelling for Impedance Based Noninvasive Diagnos-
tic Methods, Thesis for the habilitation at the Faculty of Electrical Engineering
Technical University Graz, 1999, November.
[16] M.J. Schroeder, et al., An analysis on the role of water content and state on
effective permittivity using mixing formulas, J. Biomech. Biomed. Biophys. Eng.
2 (1) (2008) 1–11.
415
[17] S. Gabriel, R.W. Lau, C. Gabriel, The dielectric properties of biological tissues. II.
Measurements in the frequency range 10 Hz to 20 GHz, Phys. Med. Biol. 41 (11)
(1996) 2271–2293.
[18] A.P. O’Rourke, et al., Dielectric properties of human normal, malignant and cir-
rhotic liver tissue: in vivo and ex vivo measurements from 0.5 to 20 GHz using
a precision open-ended coaxial probe, Phys. Med. Biol. 52 (2007) 4707–4719.
[19] A. Ferrier, et al., A microwave interferometric system for simultaneous actua-
tion and detection of single biological cells, Lab Chip 9 (23) (2009) 3406–3412.
[20] Y. Yang, et al., Distinguishing the viability of a single yeast cell with an ultra-
sensitive radio frequency sensor, Lab Chip 10 (5) (2010) 553–555.
[21] T. Chen, et al., Microwave biosensor dedicated to the dielectric spectroscopy
of a single alive biological cell in its culture medium, in: IEEE IMS Conference,
2013.
[22] C. Dalmay, et al., Ultra sensitive biosensor based on impedance spectroscopy
at microwave frequencies for single cell analysis, Sens. Actuators A: Phys. 162
(2) (2010) 189–197.
[23] C. Dalmay, et al., Microwave sensors for stem cell identification and discrim-
ination, in: IEEE MTT-S International Microwave Symposium Digest, 2010,
pp. 620–623.
[24] A. Landoulsi, et al., Tunable frequency resonant biosensors dedicated to
dielectric permittivity analysis of biological cell cytoplasm, in: IEEE MTT-S
International Microwave Symposium Digest, 2013.
[25] L. Zhang, et al., Label-free colorectal cancer cell line bio-sensing using RF res-
onator, in: Transducers-Eurosensors Conference, 2013.
[26] R. Whatley, et al., CMOS based Tunable Matching Networks for cellular handset
applications, in: IEEE MTT-S International Microwave Symposium Digest, 2011,
1, 4.
[27] S.P. Natarajan, et al., CMOS integrated digital RF MEMS capacitors, in: IEEE 11th
Topical Meeting on Silicon Monolithic Integrated Circuits in RF Systems (SiRF),
2011, pp. 173–176.
[28] L. Paavilainen, et al., The impact of tissue fixatives on morphology and antibody-
based protein profiling in tissues and cells, J. Histochem. Cytochem. 58 (2010)
237–246.
[29] J.N. Russell, J.E. Clements, L. Gama, Quantitation of gene expression in
formaldehyde-fixed and fluorescence-activated sorted cells, PLOS ONE 8 (9)
(2013) e73849.
[30] R. Buchner, et al., The dielectric relaxation of water between 0 ◦ C and 35 ◦ C,
Chem. Phys. Lett. 306 (1999) 57–63.
[31] D.L. Trainer, et al., Biological characterization and oncogene expression in
human colorectal carcinoma cell lines, Int. J. Cancer 41 (February (2)) (1988)
287–296.
[32] P. Noguchi, et al., Characterization of WiDr: a human colon carcinoma cell line,
In Vitro 15 (June) (1979) 401–408.
[33] T.U. Semple, et al., Tumor and lymphoid cell lines from a patient with carcinoma
of the colon for a cytotoxicity model, Cancer Res. 38 (May) (1978) 1345–1355.
[34] S.J. Vermeulen, et al., Did the four human cancer cell lines DLD-1, HCT-15,
HCT-8, and HRT-18 originate from one and the same patient? Cancer Genet.
Cytogenet. 107 (1998) 76–79.
[35] K. Grenier, et al., Recent advances in microwave-based dielectric spectroscopy
at the cellular level for cancer investigations, IEEE Trans. Microw. Theory Tech.
61 (May (5)) (2013) 2023–2030.
[36] D. Berube, et al., A comparative study of four open-ended coaxial probe mod-
els for permittivity measurements of lossy dielectric/biological materials at
microwave frequencies, IEEE Trans. Microw. Theory Tech. 44 (October (10))
(1996) 1928–1934.
[37] M.A. Stuchly, et al., Measurement of radio frequency permittivity of biological
tissues with an open-ended coaxial line: Part II: Experimental results, Trans.
Microw. Theory Tech. MTT-30 (1) (1982) 87–91.
[38] K. Nörtemann, et al., Dielectric properties of aqueous NaCl solutions at
microwave frequencies, J. Phys. Chem. A 101 (37) (1997) 6864–6869.
[39] A. Vander Vorst, et al., RF/Microwave Interaction with Biological Tissues, IEEE
Press/Wiley-Intersciences, 2006.
[40] G.H. Markx, et al., The dielectric properties of biological cells at radiofrequen-
cies: applications in biotechnology, Enzyme Microb. Technol. 25 (August (3–5))
(1999) 161–171.
Biographies
L.Y. Zhang (M’76–SM’81–F’87) was born on April 23, 1980, in Benxi, China. He
received the M.S. degree in electronic science from the University of the Littoral
Opal Coast, Calais, France, and the Ph.D. degree in electronics from the Univer-
sity of Western Brittany, Brest, France, in 2009. From 2010 to 2013, he has been
a postdoctoral associate at the University of Limoges, Limoges, France. His research
interests are focused on agility in microwave using ferroelectric thin films and on
characterization of microwave properties of dielectric materials.
C. Bounaix Morand du Puch was born in 1981. He received his BSc in molecular
biology from the College of Charleston (Charleston, SC, USA) and both his MSc in
biotechnology and his Ph.D. (2010) in biochemistry from the University of Greno-
ble (Grenoble, France). He has worked in academic institutions and independent
biotechnology laboratories in USA, Switzerland and France, and currently holds
a project manager position at Oncomedics (Limoges, France), developing cancer
primary models and techniques for ex vivo studies (chemotherapy sensitivity and
resistance assays, drug candidate efficacy). His past and current research interests
include venomics as well as damaged-DNA interactomes and repair mechanisms,
416 L.Y. Zhang et al. / Sensors and Actuators A 216 (2014) 405–416
involving methods of cell and molecular biology, analytical chemistry (mass spec-
trometry) and biophysics (SPR).
C. Dalmay received the Ph.D. degree in electrical engineering from the University of
Limoges, Limoges, France in 2009. She has hold a post doctoral position with the Cen-
tre National de la Recherche Scientifique (CNRS), SATIE–BIOMIS, IFR d’Alembert, ENS
Cachan working on the conception of microfluidic biochips for cell electroporation
and cell handling. She has currently an assistant professor position at the University
of Limoges, XLIM laboratory. Her main area of activity involves the development of
biodevices for cell analysis.
A. Lacroix was born on November 7, 1979. She received her BSc in biochemistry from
the University of Limoges (Limoges, France) and her MSc in infectiology from the
University of Tours (Tours, France). She obtained her Ph.D. in Virology and Cancerol-
ogy (2008) from the University of Limoges. After holding a teaching and research
(notably in glycoproteomics) assistant position (2008–2010), her current research
since 2010 as a postdoctoral associate concerns isolation and characterization of
cancer stem cells on glioblastoma model.
A. Landoulsi was born in Tunis, Tunisia, in 1987. He received the Licence degree
from the University of Limoges, France, in 2010, and the Master degree in Sci-
ences and Technologies of information and communication, specialty electronics,
optics, telecommunications from the Limoges University, France, in 2012. In October
2012, he joined the XLIM-UMR 7252 laboratory, France, where started his Ph.D. in
the research and development of RF resonant sensors for the characterization of
materials at the micro and nano scale.
J. Leroy was born on January 1, 1987. He received the Ph.D. degree in electrical engi-
neering from the University of Limoges, France in 2013. Since November 2013, he
is a postdoctoral associate at the XLIM Institute, Limoges, France. His main area of
activity is focused on the characterization of cancer cells using microwave biosen-
sors.
C. Mélin was born on October 8, 1985. She received the Ph.D. degree in Biology
specialized in cancer stem cells from the University of Limoges, France, in 2012.
After holding a teaching and research assistant position (2012–2013), her current
research since November 2013 as a postdoctoral associate concerns characterization
of colorectal cells using microwave biosensors.
F. Lalloué received the Ph.D. degree in Biology and health Sciences from the Uni-
versity of Limoges, Limoges, France in 2001. He is currently a full-time assistant
professor at the University of Limoges in the department of Physiology and is lead-
ing research into the homeostasis cellular and pathologies team. His current research
activity is focused on Neurobiology and oncology such as normal neural stem cells
and tumor stem cells in glioblastoma.
S. Battu was born in 1965. He was pharmacist and received the Ph.D. and Habili-
tation degrees from the University of Limoges, Limoges, France, in 1997 and 2003
respectively. He is currently a full-time Assistant-professor at the University of Limo-
ges, Faculty of Pharmacy, in the department of Analytical Chemistry and is leading
research into the EA 3842 “Homeostasis cellular and pathologies” team. His cur-
rent research activity is focused on cancer cells and cancer stem cells sorting by
Sedimentation Field Flow Fractionation.
C. Lautrette is CEO of Oncomedics since its creation in 2006. He received his Ph.D.
in biology and health from the University of Limoges (Limoges, France)in 2003.
After holding a teaching and research assistant position, he joined the Limousin
entrepreneur ship incubator to develop the Oncogramme, an innovative technology
dedicated to individualized cancer treatment profiling. In parallel, he also followed
the HEC Paris start-up institute program, a HEC School of management dedicated
to the creation and development of innovative business. Dr. Lautrette has authored
several research articles and is the main inventor of the Oncogramme patent. Dr.
Lautrette is co-founder of Oncomedics.
S. Giraud received her Ph.D. in biology and health from the University of Limoges
(Limoges, France) in 2005. After holding a teaching and research assistant position,
she worked for 6 months as a clinical research associate, and in parallel she also
helped developing the Oncogramme, an innovative technology dedicated to indi-
vidualized cancer treatment profiling. She is co-founder of Oncomedics and has
been CSO of the company since its creation in 2006.
A. Bessaudou received the Habilitation degree from the University of Limoges, Limo-
ges, France in 2001. She is currently a Professor at the University of Limoges, and
is leading research into XLIM research institute. Her current research activity is
focused on innovating materials elaboration and more especially metal-insulator
transition oxides.
P. Blondy (M’00) received the Ph.D. and Habilitation degrees from the University of
Limoges, Limoges, France, in 1998 and 2003 respectively. From 1998 to 2006, he was
with the Centre National de la Recherche Scientifique (CNRS), as a Research Engineer
with XLIM Laboratory, where he began research on RF-MEMS technology and its
applications to microwave circuits. He is currently a Professor at the University of
Limoges, where he holds an Institut Universitaire de France research chair. He was
a visiting researcher at the University of Michigan, Ann Arbor, USA in 1997, and at
the University of California at San Diego, La Jolla, USA in 2006 and 2008. He was an
Associate Editor for the IEEE Microwave and Wireless Components Letters in 2006.
He is a member of the IEEE International Microwave Conference Technical Program
Committee since 2003, and chair of the Technical Committee 21 on RF-MEMS. He
received the “Outstanding Young Engineer Award” from the IEEE-MTT society in
2010. He has authored or co-authored more than 150 papers in refereed journals
and conferences, holds 4 patents.
M.O. Jauberteau is Medical Doctor (University of Limoges) and Ph.D. (University
of Paris VI) and now Medical professor of Immunology (since 2000 at Uni-
versity of Limoges, Faculty of Medicine). She is the director of the Research
Team EA 3842, Homeostasis cellular and Pathologies, (HCP) created in 2004. The
research is focused on Oncology, especially mechanisms of cell survival and cancer
stem cells.
A. Pothier received the Ph.D. degree in electrical engineering from the University
of Limoges, Limoges, France in 2003. He is currently a full-time researcher with the
Centre National de la Recherche Scientifique (CNRS), XLIM, University of Limoges.
His current research activity is focused on both RF-MEMS components application
for analog communication modules and RF biosensors developments for cell anal-
ysis. He is a member of the IEEE International Microwave Conference Technical
Program Committee since 2011 and co-chair the IEEE MTT-S Technical Committee
on Biological Effect and Medical Applications of RF and Microwave since 2013. He has
authored or co-authored more than 90 papers in refereed journals and conferences,
holds 2 patents.