LABEL FREE BIO SENSING METHOD USING RADIO

FREQUENCIES SPECTROSCOPY FOR CELL DETECTION AND

DISCRIMINATION

Claire Dalmay, Arnaud Pothier, Pierre Blondy

XLIM – UMR 6172 Université de Limoges/CNRS, France

Fabrice Lalloue, Marie-Odile Jauberteau

Homéostasie cellulaire et Pathologies, Université de Limoges, France

Keywords: Bio sensor, Electrical bio-impedance, Microelectronics, RF planar devices.

Abstract: This paper presents an original label free bio sensing method allowing the study of electrical properties of

human cells and so potentially cell identification and discrimination. The proposed bio sensor is based on a

planar resonator operating at microwave frequencies, fabricated using a standard microelectronic process.

As the result its microscopic sensitive areas allow an improved detection at the cell scale which represents a

significant step in the study of many biological phenomenon. Thanks to a specific experimental protocol, we

present in this paper a simple method allowing electrical parameters measurement on a small number of

cells with a good accuracy.

1 INTRODUCTION

In recent years, biosensors known a great interest as

there is an important need for tools that can quickly

and accurately analyse biological elements like bio

molecules or cells. Current optical and chemical bio

detection techniques can effectively analyse

biological systems but present some drawbacks ;

especially their requirement of specific labels to

enhance the signal generation. These labelled

methods make the sample preparation more

complex, expensive and time consuming. In

addition, the sample can be largely chemically

altered prior analysis. In the other way, electronic

detection techniques are very interesting methods as

they allow the development of label free methods

(Kim et al. 2007). Thanks to microelectronic

technology a significant improvement of electrical

sensor detection performance can be expected since

resulting miniaturized biosensors are now able to

work at the cell scale.

In this paper, is presented an electric label free

method allowing to evaluate cell inside medium

permittivity and conductivity in the gigahertz

frequency domain. Actually, these two specific

parameters are influenced by the cell type and

morphology but also by their physiological state.

Culture chamber

Substrate (fused silica)

CPW access for

on wafer RF

measurements

Hydrophobic ring

Analysed cells

Figure 1: Schematic of the studied biosensor.

As example, tumorous cells are well known to

present a larger conductivity and permittivity than

normal cells (Blad and Baldetorp, 1996). Hence,

individual cell electrical properties measurement

represents a complementary tool allowing efficient

cell identification.

Developed biosensors are actually based on a

coplanar microwave resonator design (figure 1) able

to operate at radio frequencies with a significant

Dalmay C., Pothier A., Blondy P., Lalloue F. and Jauberteau M. (2009).

LABEL FREE BIO SENSING METHOD USING RADIO FREQUENCIES SPECTROSCOPY FOR CELL DETECTION AND DISCRIMINATION.

In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 3-6

DOI: 10.5220/0001122800030006

 SciTePress

sensitivity to tiny concentrations of biological

medium that interacts with the sensor. Moreover its

planar configuration will make easier a coming

integration in microsystems with microfluidic flow-

through network enabling accurate cell sorting

applications as example.

2 BIO DETECTION METHOD

2.1 Biosensor Design

In this study a resonant structure has been favoured

because by nature much more sensitive to very small

cell concentration in comparison with wide band

device (

Denef et al, 2004). But in the other way,

available analysis spectrum will be limited to a

narrow band around the sensor resonant frequency.

Wider band investigation will so require fabricating

several resonators with different resonant

frequencies.

Meandered inductor

Interdigital capacitor

Meandered inductor

Interdigital capacitor

Figure 2: RF Electromagnetic field distribution plot at

resonance frequency.

The developed micro biosensor has been

designed as a coplanar RLC resonator made with a

meandered inductor associated in parallel with an

inter-digital capacitor. In our case, used resonators

present a RF signal attenuation which becomes

maximum at the resonant frequency. As shown on

figure 2 at this frequency, the electromagnetic field

distribution is strongly concentrate in the capacitive

part of the device which represents the more

interesting interaction area for a capacitive detection.

Indeed, the introduction of any biological media

even in very small concentration will meaningfully

disturb the EM field distribution inducing a

detectable shift in the measured resonance frequency

of the sensor (figure 3). This frequency shift will be

all the more significant if cells are located close to

gaps between metallic lines where the

electromagnetic field is strong.

Frequency (GHz)

(dB)

Unlooaded sensor

Sensor loaded by 4 cells

Sensor loaded by 6 cells

Sensor loaded by 8 cells

Cell number

increasing

Figure 3: Electromagnetic simulation of the cell number

influence on the sensor RF response:S

parameter relies

on the RF signal attenuation through the resonator.

Hence, the detection resonator performance

strongly relies on the biosensor sensitivity

capabilities where two parameters play a major role.

First, interaction between the EM field and cells to

be analysed must be maximized using an

appropriated sensor design with gaps between

metallic lines in the same order of magnitude of

analysed cell sizes: in the present case considered

gaps will be close to 10 µm. Then, a sufficient

resonator unloaded quality factor (relative the

resonator intrinsic loss) has to be also considered; as

it controls how the resonant frequency pick will be

narrow and so the sensor frequency sensitivity to a

small frequency shift.

Once biological cells will be present on the

sensor surface, both their location and their number

will directly influence its response. As shown on

figure 3, following our approach the detection of a

low number of cells (at least less than ten) can be

expected.

In the end, a microfluidic network will certainly

be required but in order to demonstrate the sensor

capability, we have chosen to develop a specific

experimental protocol for instance, allowing an

easier test procedure with the cost of the difficulty to

work with real alive cells. This protocol will be

presented in the following paragraphs.

2.2 Biosensor Fabrication Process

Micro sensors are fabricated using standard

microelectronic process with biocompatible

materials (figure 4).

BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices

Figure 4: SEM photograph of the fabricated micro sensor.

A fused silica substrate has been preferred to

classical silicon one especially for its lower loss

properties in the RF frequency domain and also for

its transparency that makes easier the observation of

cells throw it. A classical photolithography allows to

define thin gold lines which are next electroplating

up to a thickness of 3 µm. Then a SU8 photoresist

from Microchem is used to create a 20µm thick well

localised culture micro-chamber on the sensor

surface.

2.3 Experimental Protocol

During characterizations, we have to ensure the

integrity of cells. Usually, a support biological

media, in which cells could be protected, is required.

As shown on figure 5, most of support medium

commonly used in biology are aqueous saline

solutions which present very strong losses for RF

signal especially when the sensor is fully cover with

it. Actually in this configuration, the biological

media alters in a too important manner the RF

performance of the device avoiding any accurate

detection.

5 10152025303504

-25

-20

-15

-10

-5

-30

1: Empty sensor

3: Distilledwater

2: PBS

4: Culture media

(GHz)

Biological

me di a

deposition

Drop of biological media

Frequency

(dB)

5 10152025303504

-25

-20

-15

-10

-5

-30

1: Empty sensor

3: Distilledwater

2: PBS

4: Culture media

(GHz)

Biological

me di a

deposition

Drop of biological media

Frequency

(dB)

Figure 5: Influence of different biological support media

20nl drops on the sensor RF performances.

A first solution could be to limit used biological

media to a very small volume, typically implying a

microfluidic flow-through system.

Another alternative will be to perform analysis in

a specific low loss support media, as done in

previous work (Dalmay et al., 2008) using ficoll (a

polymeric gel of sucrose). Hence, once dried, the

ficoll drop allows to protect cells to be analysed in a

low permittivity polymeric matrix with the cost of a

aleatory cell location on the sensor surface and in the

ficoll matrix that induces a significant error in their

electrical parameter extraction. Consequently, there

is a great interest in developing an experimental

protocol without any biological support media.

Actually with the proposed approach in this

paper, cells are directly grown on the sensor surface

submerged in a classical culture media. Few days are

required to allow a sufficient cell adhesion on the

sensor surface. Then sensor are washed in deionised

water following by paraformaldehyde 4 % bath

(PFA) in order to definitively fixe cells and so to

avoid cell degradation during the measurement

sequence. Since most of cell adhesion occurs

preferentially on the silica substrate than on gold

lines or on SU8 resist, most of fixed cells are located

only between gold lines in the culture micro-

chamber (figure 1). Number of cell on the sensor are

roughly controlled both with the cell concentration

initially dispersed in culture media and the culture

time.

After 5 days in

the culture

medi a

10 min

PFA4% with

deionised water

Sensor drying

Adherent cells

floating cells

Biosensor

After 5 days in

the culture

medi a

10 min

PFA4% with

deionised water

Sensor drying

Adherent cellsAdherent cells

floating cells

BiosensorBiosensor

Figure 6: Experimental protocol process.

After PFA bath, cells are no longer alive, but

their original form, their intracellular content and

their electrical properties have been kept as in living

conditions. Then sensor are again washed in

deionised water and dried just before measurement.

LABEL FREE BIO SENSING METHOD USING RADIO FREQUENCIES SPECTROSCOPY FOR CELL DETECTION

AND DISCRIMINATION

3 EXPERIMENTAL RESULTS

All characterizations are performed using classical

microwave measurement techniques with on wafer

probing; as it allows a quick and a successive sensor

measurements.

First, for each sensor, the transmitted microwave

signal attenuation across the unloaded resonator is

recorded using a calibrated vector network analyser.

Then cell culture is performed; at the end, loaded

sensors with fixed cells are measured following the

same procedure than before cell growth. Hence, the

induced resonator frequency shift value, related to

the cells electrical properties can be extracted.

Frequency (GHz)

(dB)

Unloaded sensor

EM simulation

Measurement

Sensor after cell growth

370MHz

Figure 7: Biosensor measured response before and after

the cell growth and simulated one.

Figure 7 shows results of experimentations with

glial-cells-derived tumour glioblastoma coming

from human nervous system cells. Used biosensors

initially resonate at 16 GHz and shift down to 15.63

GHz when it is loaded with only 8 glial-cells (figure

8).

Figure 8: Photograph of the sensor after the cell growth.

Fullwave simulations, based on finite element

method (HFSS from ANSOFT), are then used to

extract individual cell electrical properties. Cells EM

modelling is done assuming that they are

homogenous, source-free and linear dielectric

volume. Hence, on a narrow frequency bandwidth

around the sensor resonant frequency, cell global

permittivity and conductivity can be extracted with a

good accuracy by fitting simulations data with

measured one, as shown on figure 7.

Hence in the case of analysed glial-cells, we

have obtained an effective permittivity value of 36 ±

1 while global conductivity has been estimated

0.100 ± 0.003 S/m at 16 GHz and 20°C. These

results agree very well with previous analysis done

with ficoll media (Dalmay et al., 2008) and can be

compared to the effective permittivity of pure water

which is closed to 45 at 20°C. Other

characterizations are currently done with other

cellular types, to demonstrate that it is possible with

this approach to discriminate between different cell

types.

4 CONCLUSIONS

An original label free bio-sensing approach for

cellular analysis at radio frequencies has been

demonstrated. Thanks to their sub millimetric size,

used sensors are able to work at the cell scale with a

very limited number of cells and can potentially be a

novel promising tool for cell discrimination. Further

work is ongoing to evaluate experimentally the

minimum number of cell analysis achievable and to

improve the sensor design and experimental process

for one single cell analysis.

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Young-Il Kim, Yunkwon Park, Hong Koo Baik, 2007.

“Development of LC resonator for label-free

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T. W. Athey, M. A. Stuchly, S. S. Stuchly, 1982.

“Measurement of radio frequency permittivity of

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BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices