Fiber Optic Biosensor Adapted to Cell and Tissue Culture

Situations for Detection of Membrane Receptors

“The Gap Step towards Non-Invasive Clinical Biosensing” Viera Malachovská1,2, Clotilde Ribaut1,2, Valérie Voisin1, Patrice Mégret1, Ruddy Wattiez2, Christophe Caucheteur1

1University of Mons (UMONS), Faculty of Engineering, Electromagnetism and Telecommunication Department, Boulevard Dolez 31, Mons, Belgium 2University of Mons (UMONS), Institute for Bioscience, Proteomics and Microbiology Laboratory, Avenue du Champ de Mars 6, Mons, Belgium

Matinée des Chercheurs 2013 | 12 Mars 2013 viera.malachovska@umons.ac.be

Introduction The ability of examining living cells is crucial to cell biology. Biosensors, as a class of analytical

instruments can provide real-time quantitative information on the interaction on the level of single

proteins, and therefore allow us to study cell processes such as cell signaling, cell communication

and cell adhesion. However, most of this label-free devices are based on the direct contact of the

cell to the device. In other words, the cells are forced in growth onto the device surface. Likewise,

processes concerning the interactions with membrane receptors are important for many clinical

studies, even though due to many technical reasons theses studied are limited to conditions

where the receptor is still in its lipid environment. Therefore, bioscience is in need for label-free

biosensor which would allow to study the membrane receptor interactions in in vitro conditions.

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Figure 1. Comparison of two work transmission spectra depending on the surface modification. The

bare tilted fiber Bragg grating spectra (TFBG; left bottom picture). TFBG spectra after coated with a

nanolayer of gold (AuIII) nanoparticles (right bottom picture) with surface Plasmon resonance (SPR)

signature. Top images are surface topography scanned by atomic force microscope (AFM) and

here are presented as 500 x 500 nm2 AFM 3D images of bare glass optical fiber (top left), the fiber

with gold coating (right top). The vertical scale is 30 nm for all images. The measurements are

carried out in air with PeakForce Tapping mode.

SPR signature

125 µm

20 nm

6 nm

Figure 3. Schematic illustration of the lateral view on the OF

biosensor based on TFBG-SPR probe with immobilized

antibodies (Abs, red - right zoom) through alkanethiolates

(ATs, grey) on gold (yellow). Top left picture is a 500 x 500

nm2 AFM 3D image of Abs on ATs creating a biofilm. The

vertical scale is 30 nm. The measurements are carried out

in air with PeakForce Tapping mode.

Figure 2. Schematic representation of the fiber optic sensor working setup; polarization

controller (PC) and, computer-Optical Vector Analyzer (OVA LUNA), optical fiber (OF) and

fiber optic sensor region (TFBG-SPR). Top zoom image is of common single mode glass OF

with polymer coating. Bottom zoom image is of TFBG-SPR. This sensor part has a in-core

inscribed grating which is 1 cm long with 7° internal tilt. The “greenish” colour is due to the 40

nm gold coating deposited in two equal steps by means of magnetic sputtering process in air

(see material and methods).

Acknowledgement for Collaborations Mathieu Surin, Philippe Leclère, University of Mons, Faculty of Sciences,

Laboratory for Chemistry of Novel Materials, Mons, Belgium.

Prof. Alexandra Belayew, Armelle Wauters, Laboratory of Molecular Biology,

Mons, Belgium.

Dr. Normando Enrique Iznaga-Escobar, Center of Molecular Immunology

CIMAB SA, Havana, Cuba.

Prof.dr.G.P.M. Luyten, Martine J. Jager, Mieke Versluis, Department of

Ophthalmology, Leiden, Netherlands.

State of Art In our case study, the fiber-optic biosensor is used as a direct in vitro non-invasive and label-free optical biosensor platform

for real-time detection of extracellular membrane receptors in alive cells. Tilted fiber Bragg grating surface Plasmon

resonance (TFBG-SPR) assay sensor was used as a transducer, able to differentiate between

mechanical/chemical/temperature cross-sensitivities. The sensor was biofunctionalized through common carboxy-

alkanethiolates/amin-coupling/antibody covalent immobilization strategy. As a result, this immunosensor can be used to

target and detect extracellular membrane receptors in native membranes of different human epithelial cell lines through the

specific affinity interaction with it’s surface immobilized antibodies (Ab). In this work the effectiveness of the presented device

is studied on a cell culture grown in a monolayer and in cell suspensions. We have chosen two model systems for this study

of interactions, a cell line with overexpressed membrane receptors (a positive control) and cell line without these receptors (a

negative control).

Summary This study in now only in the process of investigation. Therefore, no results

concerning the kinetic of the receptor interaction are presented. This study is

based on the assumption, that presented optical biosensor technology can

differentiate between the high specific affinity membrane receptor interactions

and the physical contact with the cells in real time. The complexity of the

interactions is left for the discussion.

TFBG-SPR sensor is able to sense cells

which are present in the sensing region

and in the penetration field. Cells present

opposite to the sensing field (blue) are not

detected.

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PC

Computer

OVA-LUNA

Figure 6. Illustration of the interaction between extracellular cell

membrane receptors and antibodies immobilized to the sensor surface (Images source; www.biooncology.com, www.123rf.com/photo_13696208_chemical-structure-of-an-immunoglobulin-g-igg-antibody).

0.5 mm

Figure 4. Image of TFBG-SPR probe placed onto

a culture of cells grown in a monolayer (with

a confluence of 90%), magnification 4 x 10.

Figure 5. Image of TFBG-SPR probe immersed

into a suspension of cells (spherical shapes),

magnification 4 x 10.

A B

Penetration depth is exponentially decaying

from the sensor into the surrounding

medium at minimum sensitivity around

200 nm. In case “A” the sensor senses only

the cells present towards the sensing field.

The interaction between the biosensor and cell is reversible. However the time (t) of interaction vary.

tA tB

Two cell culture situations:

monolayer of cells (A, left), cell suspension (B)

Figure 7. Sensogram of TFBG-SPR sensor. Below a schematic

image of the reflected beam from the inscribed tilted grating.

Buffer

Cell culture