Biosensor technique based on

Optical Waveguide Lightmode

Spectroscopy - OWLS

www.owls-sensors.com

 Copyright 2004
2004, MicroVacuum Ltd. All Rights Reserved.
Reproduction, reprint or any other form of reuse of the whole presentation document
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© 2007 MicroVacuum
MicroVacuum Ltd.
Hungarian owned company established in 1987.
Main activity:
• research, development in thin film and sensor technology,
• production of biosensors and related instruments.
MicroVacuum is involved in international and national research
projects in the field of biosensors and nanotechnology.
The company has ISO 9001 certification from 1997.

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Content
• Introduction to OWLS technique
• Applications
• OWLS 120 instrument and OW sensors
• OWLS sensing theory
• Experimental procedure

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Introduction of OWLS biosensing technique
Optical grating coupler waveguide sensor
• Planar optical waveguide on glass substrate
• Grating incorporated in planar waveguide
• Light intensity spectrum of the incoupled, guided light mode (TE,TM)
• Surface of the waveguide is exposed with solution containing molecules
• The spectrum shifts due to molecular interactions with the surface

Main features
• Highly sensitive technique
• Real-time monitoring of surface
interactions
• No labeling required
• Absolute number of adsorbed
molecules can be determined
• Conformational change information
can be deduced

© 2007 MicroVacuum
Main Biosensing Applications of OWLS
more details in www. owls-sensors.com
• Ligand/receptor binding
• Immunosensing (antibody/antigen)
• Lipid bilayers - biomembranes
• Protein - DNA interactions
• Biomaterials –interaction of surface with blood plasma, serum
and proteins
• Interactions with cells – toxicology, cell-surface interactions
• Monitoring environmental pollution
• Analysis of association and dissociation kinetics
• Kinetics of adhesion, growth and spreading of living cells
• Molecular self-assembly & nanoscience

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OWLS 120
Optical Waveguide Light-mode Spectroscopy System

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Measuring Unit
• Optical-mechanical system
• Electronic signal processing and controlling unit
• Industrial PC based computer with
hard disk, CD reader, USB flash memory
• Integrated sensor holder
• Temperature control subunit
• Sample injection subunit

External monitor, keyboard and mouse

are connected on the back panel of the Measuring Unit.

BioSense software
Microsoft Windows® XP based software provides flexible control of the
measurement, data evaluation, analysis and storage.

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OWLS Optical-mechanical system
schematic drawing
rotator

flowcell

waveguid substrate
e
photodiode

shutter laser
beam mirror

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OW 2400
optical grating coupler waveguide sensor

Grating Periodicity: 2400 lines/mm Waveguide layer

Depth: 10-20 nm Thickness 200 nm
Grating width (l) 2 mm H
W
L
Gra ting I
Dot mark
F(wa vegu ide film)
Sub(s strate ssgla )

Refractive index 1.8

Typically: SixTi1-xO2
Grating I x=0.25± 0.05
Dot mark

L
Glass substrate
Length 12 mm
F (waveguide layer)
Width 8 mm
S (glass substrate)
H Thickness 0.55 mm
W Refractive index 1.53

Inorganic/organic coatings are available on request.

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 Surface modifications

Inorganic coating:
• SiO2, TiO2, Ta2O5, ZrO2, Nb2O5, Al2O3

• Electrochemical/optical sensor: ITO

Organic/biofunctional coating:
• Teflon ®
• amino groups
• epoxy
• carboxylate
• biotinated surface

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 OWLS sensing theory
Optical grating coupler waveguide sensor model

nC cover medium (C)

nA, dA added layer (A)
nF, dF waveguide layer (F)
nS
glass substrate (S)
laser light

C ∆ Z
Evanescent field
A ∆ Z ≈ 200 nm
EM distribution
F

S © 2007
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1 MicroVacuum Ltd.
 Basic calculation procedure
Incoupling condition

The incoupling angles α( TE), α

(TM) of electric and magnetic
modes are evaluated from the
measured lightmode spectra.

The effective refractive kλ

indices N(TE), N(TM) of the NTE = nair sin(α TE ) +
waveguide Λ
structure are calculated on
the basis of incoupling kλ
condition. NTM = nair sin(α TM ) +
Λ
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 Waveguiding mode conditions
Mode equations for four-layer waveguide structure

   N 2  N 2 
ρ 
 
    +   − 1  
2 n2A − n2   nC   nA   
2
0 = k nF − N dF + 2

C
  dA  − Two equations:
nF − n2C  N 
2
 N
2

   +   − 1  N= NTE and ρ =0 for TE
 n  n   
  C   F  
N= NTM and ρ =1 for TM

 n 2ρ N2 − n2   n 2ρ N2 − n2 
C S
− arctan F   − arctan F  
 n C  n2 2   n S  2 2 
 F −N   nF − N 

Two independent parameters can be calculated

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 Calculation of nF and dF
The waveguide layer parameters nF and dF are calculated from the
mode equations providing that dA=0 - three-layer model.

Calculation of nA , dA and M
Sensor with added layer – four-layer model
The refractive index (nA) and the thickness (dA) of the added layer
are calculated.

Using the model that the refractive index in the adsorbed layer
linearly depends on the concentration of the adsorbed material,
the mass per unit area (M) of the adsorbed material
can be calculated.
n − nC For most protein adsorption
M = dA A dn/dc = 0,182 cm3/g
dn / dc

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 OWLS spectrum – Peak measurement

Incoupled light intensity

versus angle of incidence
of the laser light

NTE(α TE), NTM(α TM) α TE

nF(NTE, NTM, nC, nS) α TM

dF(NTE, NTM, nC, nS) FWHM

nA(NTE, NTM, nC, nS, nF , dF)

dA(NTE, NTM, nC, nS, nF , dF)

M (nA, dA, dn/dc)

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 Monitoring surface processes – RIM measurement
• Repeating the Optical Waveguide Light-mode Spectrum measurements
• Time-dependence of the optical parameters are presented on graph
and analyzed.
NTE(t), NTM(t),

nA(t), dA(t), M(t)

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© 2007 MicroVacuum
 A typical example of an OWLS experiment using proteins
Baseline-run in pure (non-film
forming) buffer : determination
of the refractive index nF and the 250
buffer

Absorbed mass of proteins

thickness, dF, of the waveguide .
Eliminates small differences in
200
sensor parameters from one 0.4mg/ml
chip to other. (ng/cm2) BSA
Adsorption experiment monitors 150
the evolution of nA, dA and M. In Shift of the
situ determination of adsorbed incoupling angles
100
mass and the kinetics of adlayer
formation.
50
Desorption phase: this is a Baseline
washing step in pure buffer, in buffer
which provides information 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130
about the stability of the
time (min)
formed adlayer and possible
desorption kinetics.

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 Sensitization of the waveguide sensors with monoclonal anti-HSP70 antibodies

GA - injection of glutaraldehyde
600
(2.5% in distilled water) to activate HCl HCl
amino groups, 500
TRIS - changing distilled water to

Mass (ng/cm2)
tris buffer (42mM, pH 7.4), 400
Blocking
MoAb - injection of monoclonal 300
anti-HSP70 IgG solution (33µ g/ml MoAb
in tris buffer) to form the sensitive 200
layer, GA
100
HCl - injection of hydrochloric acid TRIS
(10mM) to wash the surface, 0
Blocking - blocking free reactive 0 20 40 60 80 100 120 140
aldehyde groups with glycine Time (min)
(10mM in tris buffer)

The experiment was performed with a FIA system with injection loop volume of 200 µl and with flow rate of 90 µl/min

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 HSP70 protein binding to immobilized anti-HSP70 antibodies

500
10 pg/ml 20 pg/ml
5 pg/ml
1 pg/ml

sample
HSP70 450

Sensor response (a.u.) xx

monoclonal antibody

100 - 150 nm 400

laser light waveguide

EM field glass 350

distribution support

300

250
The lowest detectable amount of HSP70
is 1 pg/ml. 100 150 200 250 300
Time (min)

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 Model experiment performed on amino functionalized surface
Coverage (ng/cm2)
600
5
anti-BSA IgG r
500 4 r
3 r
r 1 r 2 r
400
BSA

300

200 A - distilled water 1 - 10µg/ml IgG

B - 2.5% glutaraldehyde 2 - 25µg/ml IgG
D C - TRIS buffer, pH=7.4 3 - 50µg/ml IgG
100 D - 200 µg/ml BSA (TRIS) 4 - 100µg/ml IgG
B
C r - regeneration solution, 0.1 M HCl 5 - 200µg/ml IgG
0
0 20 40 60 80 100 120
A
Time (min)
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 Parameters that OWLS can measure
-6
Parameter Sensitivity (*10 ) Known parameters Possible use
nF 700 000 nC, nS, λ, Λ gas detection, swelling
-1
dF 860 nm nC, nS, λ, Λ gas detection, swelling

nC 99000 dF, nF, nS, λ, Λ cells, concentration

-1
dA 210 nm dF, nF, nC, nS, λ, Λ SAM, protein size
pH 50 calibration needed pH meter
-1
T 10 °C calibration needed -
2
Mass (protein) 300 cm /µg dF, nF, nC, nS, λ, Λ adsorption, binding
2
Mass (lipids) 150 cm /µg dF, nF, nC, nS, λ, Λ adsorption, binding
-1
p 50 (1000) bar calibration needed pressure meter
flow rate 8000 h/ml calibration needed flow rate meter

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 Non-biosensing applications of
the optical grating-coupler waveguide sensor
• Incoupling laser light for total internal reflection
• Incoupling laser light for Raman spectroscopy
• Incoupling laser light for optical loss measurement in
thin films
• Incoupling laser light for SPR measurement
• Incoupling laser light for fluorescent excitation
• In situ monitoring and characterization of nanometer scale
thin layers
• Humidity and gas monitoring

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 Thank you for your kind attention

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