Biosensor technique based on

Biosensor technique based on
Optical Waveguide Lightmode
Optical Waveguide Lightmode
Spectroscopy - OWLS
Spectroscopy - OWLS
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MicroVacuum
MicroVacuum
Ltd.
Ltd.
Hungarian owned company established in 1987. Hungarian owned company established in 1987.
Main activity: Main activity:

research, development in thin film and sensor technology, research, development in thin film and sensor technology,

production of biosensors and related instruments. production of biosensors and related instruments.
MicroVacuum is involved in MicroVacuum is involved in international and national international and national research research
projects in the field of biosensors and nanotechnology. projects in the field of biosensors and nanotechnology.
The company has ISO 9001 certification from 1997. The company has ISO 9001 certification from 1997.
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Content
Content

Introduction to OWLS technique Introduction to OWLS technique
• Applications Applications
• OWLS 1 OWLS 12 20 instrument and OW sensors 0 instrument and OW sensors
• OWLS sensing theory OWLS sensing theory
• Experimental procedure Experimental procedure
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Introduction of OWLS biosensing technique
Introduction of OWLS biosensing technique
Optical grating coupler waveguide sensor Optical grating coupler waveguide sensor
• Planar optical waveguide on glass substrate Planar optical waveguide on glass substrate
• Grating incorporated in planar waveguide Grating incorporated in planar waveguide
• Light intensity spectrum of the incoupled, guided light mode (TE,TM) Light intensity spectrum of the incoupled, guided light mode (TE,TM)
• Surface of the waveguide is exposed with solution containing molecules Surface of the waveguide is exposed with solution containing molecules
• The spectrum shifts due to molecular interactions with the surface The spectrum shifts due to molecular interactions with the surface
Main features Main features
• Highly sensitive technique Highly sensitive technique
• Real-time monitoring of surface Real-time monitoring of surface
interactions interactions
• No labeling required No labeling required
• A Absolute number of adsorbed bsolute number of adsorbed
molecules molecules can can be determined be determined
• Conformational change information Conformational change information
can be deduced can be deduced
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Main Biosensing Applications of OWLS
Main Biosensing Applications of OWLS
• Ligand/receptor binding Ligand/receptor binding
• Immunosens Immunosensi ing ng (antibody/antigen (antibody/antigen) )
• Lipid bilayers - biomembranes Lipid bilayers - biomembranes
• Protein - DNA interactions Protein - DNA interactions
• Biomaterials –interaction of surface with blood plasma, serum Biomaterials –interaction of surface with blood plasma, serum
and proteins and proteins
• Interactions with cells – toxicology, cell-surface interactions Interactions with cells – toxicology, cell-surface interactions
• Monitoring environmental pollution Monitoring environmental pollution
• Analysis of association and dissociation kinetics Analysis of association and dissociation kinetics
• Kinetics of adhesion, growth and spreading of living cells Kinetics of adhesion, growth and spreading of living cells
• Molecular self-assembly & nanoscience Molecular self-assembly & nanoscience
more details in www. more details in www. owls-sensors owls-sensors.com .com
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OWLS 1
OWLS 1
2
2
0
0
Optical Waveguide Light-mode Spectroscopy System Optical Waveguide Light-mode Spectroscopy System
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Measuring Unit Measuring Unit
• Optical-mechanical system Optical-mechanical system
• Electronic signal processing and controlling unit Electronic signal processing and controlling unit
• Industrial PC based computer with Industrial PC based computer with
hard disk, CD reader hard disk, CD reader, , USB flash memory USB flash memory
• Integrated sensor holder Integrated sensor holder
• Temperature control subunit Temperature control subunit
• Sample injection subunit Sample injection subunit
External monitor, keyboard and mouse External monitor, keyboard and mouse

are are connected on the back panel of the Measuring Unit. connected on the back panel of the Measuring Unit.
BioSense software BioSense software
Microsoft Windows Microsoft Windows
® ®
XP XP based software provides flexible control of the based software provides flexible control of the
measurement, data evaluation, analysis and storage. measurement, data evaluation, analysis and storage.
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OWLS
OWLS
Optical-mechanical system
Optical-mechanical system

schematic drawing
schematic drawing
laser laser
rotator rotator
flowcell flowcell
photodiode photodiode
waveguid waveguid
e e
substrate substrate
shutter shutter
beam mirror beam mirror
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Periodicity: 2400 lines/mm Periodicity: 2400 lines/mm
Depth: 10-20 nm Depth: 10-20 nm
Grating width (l) 2 mm Grating width (l) 2 mm
Waveguide layer Waveguide layer
Thickness 200 nm Thickness 200 nm
Refractive index 1 Refractive index 1. .8 8
Typically: Si Typically: Si
x x
Ti Ti
1-x 1-x
O O
2 2
x=0 x=0. .25 25t t 0 0. .05 05
Glass substrate Glass substrate
Length 12 mm Length 12 mm
Width 8 mm Width 8 mm
Thickness 0 Thickness 0. .55 mm 55 mm
Refractive index 1 Refractive index 1. .53 53
OW 2400
OW 2400
optical grating coupler waveguide sensor
optical grating coupler waveguide sensor
Grating Grating
© © 2007 2007 MicroVacuum MicroVacuum
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Inorganic/organic coatings are available on request. Inorganic/organic coatings are available on request.
I Dot mark F(wa vegu ide film) S(s ub strate gla ss ) Gra ting W L H
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Dot mark
F (waveguide layer)
S( substrate) glass
I
W
H
Grating
L

Inorganic coating: Inorganic coating:
• SiO SiO
2 2
, TiO , TiO
2 2
, Ta , Ta
2 2
O O
5 5
, ZrO , ZrO
2 2
, Nb , Nb
2 2
O O
5 5
, Al , Al
2 2
O O
3 3

• Electrochemical/optical sensor: ITO Electrochemical/optical sensor: ITO
Organic/biofunctional coating: Organic/biofunctional coating:
• Teflon ® Teflon ®
• amino groups amino groups
• epoxy epoxy
• carboxylate carboxylate
• biotinated surface biotinated surface
Surface modifications
Surface modifications
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cover cover medium (C) medium (C)
glass substrate (S) glass substrate (S)
waveguide layer (F) waveguide layer (F)
added layer (A) added layer (A)
n n
S S
n n
F F
, d , d
F F
n n
A A
, d , d
A A
laser light laser light
n n
C C
Optical grating coupler waveguide sensor Optical grating coupler waveguide sensor model model
EM distribution EM distribution
OWLS sensing theory
OWLS sensing theory
∆ ∆ Z Z
C C
A A
F F
S S
Evanescent field Evanescent field
∆ ∆ Z Z ≈ ≈ 200 nm 200 nm
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Basic calculation procedure
Basic calculation procedure
Incoupling condition Incoupling condition
Λ
λ
α
k
) sin( n N
TE air TE
+ ·
Λ
λ
α
k
) sin( n N
TM air TM
+ ·
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12
The effective refractive The effective refractive
indices indices N(TE), N(TM) N(TE), N(TM) of the of the
waveguide waveguide
structure structure are calculated on are calculated on
the basis of incoupling the basis of incoupling
condition. condition.
The incoupling angles The incoupling angles α( α( TE), TE), α α
(TM) (TM) of electric and magnetic of electric and magnetic
modes are evaluated from the modes are evaluated from the
measured lightmode spectra. measured lightmode spectra.
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Waveguiding mode conditions
Waveguiding mode conditions
]
]
]
]


,
`

.
|

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]
]


,
`

.
|

,
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.
|
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]
]

,
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|
+

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|

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|
+

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+ − ·
2 2
F
2
S
2
2
S
F
2 2
F
2
C
2
2
C
F
A
2
F
2
C
2
A
2
C
2
C
2
F
2
C
2
A
F
2 2
F
N n
n N
n
n
arctan
N n
n N
n
n
arctan
d
1
n
N
n
N
1
n
N
n
N
n n
n n
d N n k 0
ρ ρ
ρ

Two equations: Two equations:
N= N N= N
TE TE
and and ρ ρ=0 for TE =0 for TE
N= N N= N
TM TM
and and ρ ρ=1 for TM =1 for TM
Mode equations for four-layer waveguide structure Mode equations for four-layer waveguide structure
T Two wo independent independent parameters can be calculated parameters can be calculated
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The waveguide layer parameters n The waveguide layer parameters n
F F
and d and d
F F
are calculated from the are calculated from the
mode equations providing that dA=0 - mode equations providing that dA=0 - three-layer model. three-layer model.
Calculation of n
Calculation of n
F F
and d
and d
F F
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14
Calculation of n
Calculation of n
A A
, d
, d
A A
and M
and M
Sensor with added layer – Sensor with added layer – four-layer model four-layer model
The refractive index The refractive index (n (n
A A
) ) and the thickness and the thickness (d (d
A A
) ) of the added layer of the added layer
are calculated. are calculated.
Using the model that the refractive index in the adsorbed layer Using the model that the refractive index in the adsorbed layer
linearly linearly depends on the concentration of the adsorbed material, depends on the concentration of the adsorbed material,
the mass per unit area the mass per unit area (M) (M) of the adsorbed material of the adsorbed material
can be calculated. can be calculated.
dc / dn
n n
d M
C A
A

·
For most protein adsorption For most protein adsorption
dn/dc = 0,182 cm dn/dc = 0,182 cm
3 3
/g /g
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OWLS spectrum – Peak measurement
OWLS spectrum – Peak measurement
Incoupled light intensity Incoupled light intensity
versus angle of incidence versus angle of incidence
of the laser light of the laser light
α α
TE TE
α α
TM TM
FWHM FWHM
N N
TE TE
( (α α
TE TE
), N ), N
TM TM
( (α α
TM TM
) )
n n
F F
(N (N
TE TE
, , N N
TM TM
, n , n
C C
, n , n
S S
) )
d d
F F
(N (N
TE TE
, , N N
TM TM
, n , n
C C
, n , n
S S
) )
n n
A A
(N (N
TE TE
, , N N
TM TM
, n , n
C C
, n , n
S S
, n , n
F F
, d , d
F F
) )
d d
A A
(N (N
TE TE
, , N N
TM TM
, n , n
C C
, n , n
S S
, n , n
F F
, d , d
F F
) )
M (n M (n
A A
, ,

d d
A A
, dn/dc) , dn/dc)
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Monitoring surface processes – RIM measurement Monitoring surface processes – RIM measurement
• Repeating the Optical Waveguide Light-mode Spectrum measurements Repeating the Optical Waveguide Light-mode Spectrum measurements
• Time-dependence of the optical parameters are presented on graph Time-dependence of the optical parameters are presented on graph
and analyzed. and analyzed.
.
.
N N
TE TE
(t), N (t), N
TM TM
(t), (t),
n n
A A
(t), d (t), d
A A
(t), M(t) (t), M(t)
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16
.
.

A typical example of an OWLS experiment using proteins A typical example of an OWLS experiment using proteins
0
50
100
150
200
250
0 10 20 30 40 50 60 70 80 90 100 110 120 130
time (min)
A
b
s
o
r
b
e
d

m
a
s
s

o
f

p
r
o
t
e
i
n
s

(
n
g
/
c
m
2
)

Baseline Baseline
in buffer in buffer
Shift of the
incoupling angles
0 0. .4mg/ml 4mg/ml
BSA BSA
buffer buffer
Baseline-run Baseline-run in pure (non-film in pure (non-film
forming) buffer : determination forming) buffer : determination
of the refractive index of the refractive index n n
F F
and the and the
thickness, thickness, d d
F F
, of the waveguide . , of the waveguide .
Eliminates small differences in Eliminates small differences in
sensor parameters from one sensor parameters from one
chip to other. chip to other.
Adsorption experiment Adsorption experiment monitors monitors
the evolution the evolution o of f n n
A A
, , d d
A A
and M. In and M. In
situ determination of adsorbed situ determination of adsorbed
mass and the kinetics of adlayer mass and the kinetics of adlayer
formation. formation.
Desorption phase: Desorption phase: this is a this is a
washing step washing step i in pure buffer, n pure buffer,
which provides information which provides information
about the stability of the about the stability of the
formed adlayer and possible formed adlayer and possible
desorption desorption kinetics. kinetics.
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0
100
200
300
400
500
600
0 20 40 60 80 100 120 140
Time (min)
M
a
s
s

(
n
g
/
c
m
2
)
GA
TRIS
MoAb
HCl
Blocking
HCl
Sensitization of the waveguide sensors with monoclonal anti-HSP70 antibodies Sensitization of the waveguide sensors with monoclonal anti-HSP70 antibodies
GA GA - - injection of glutaraldehyde injection of glutaraldehyde
(2.5% in distilled water) to activate (2.5% in distilled water) to activate
amino groups, amino groups,
TRIS TRIS - changing distilled water to - changing distilled water to
tris buffer (42mM, pH 7.4), tris buffer (42mM, pH 7.4),
MoAb MoAb - injection of monoclonal - injection of monoclonal
anti-HSP70 IgG solution (33 anti-HSP70 IgG solution (33µ µg/ml g/ml
in tris buffer) to form the sensitive in tris buffer) to form the sensitive
layer, layer,
HCl HCl - injection of hydrochloric acid - injection of hydrochloric acid
(10mM) to wash the surface, (10mM) to wash the surface,
Blocking Blocking - blocking free reactive - blocking free reactive
aldehyde groups with glycine aldehyde groups with glycine
(10mM in tris buffer) (10mM in tris buffer)
18
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 HSP70 protein binding to immobilized anti-HSP70 antibodies anti-HSP70 antibodies
19
The lowest detectable amount of HSP70
is 1 pg/ml.
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250
300
350
400
450
500
100 150 200 250 300
Time (min)
S
e
n
s
o
r

r
e
s
p
o
n
s
e

(
a
.
u
.
)

x
x



1 pg/ml
5 pg/ml
10 pg/ml 20 pg/ml
waveguide
glass
support
sample
laser light
EM field
distribution
100 - 150 nm
monoclonal antibody
HSP70
waveguide
glass
support
sample
laser light
EM field
distribution
100 - 150 nm
monoclonal antibody
HSP70

Model experiment performed on amino functionali Model experiment performed on amino functionaliz zed surface ed surface
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20
0 20 40 60 80 100 120
Time (min)
0
100
200
300
400
500
600
Coverage (ng/cm2)
1
2
3
4
5
A
B
C
D
r
r
r
r
r
r
A - distilled water
B - 2.5% glutaraldehyde
C - TRIS buffer, pH=7.4
D - 200 µg/ml BSA (TRIS)
r - regeneration solution, 0.1 M HCl
1 - 10µg/ml IgG
2 - 25µg/ml IgG
3 - 50µg/ml IgG
4 - 100µg/ml IgG
5 - 200µg/ml IgG
BSA
anti-BSA IgG
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Parameters
Parameters
that
that
OWLS can
OWLS can
m
m
easure
easure
Parameter Sensitivity (*10
-6
)
Known parameters Possible use
n
F
700 000 n
C
, n
S
,
λ, Λ gas detection, swelling
d
F
860 nm
-1
n
C
, n
S
,
λ, Λ
gas detection, swelling
n
C
99000 d
F
, n
F
, n
S
,
λ, Λ
cells, concentration
d
A
210 nm
-1
d
F
, n
F
, n
C
, n
S
,
λ, Λ
SAM, protein size
pH 50 calibration needed pH meter
T 10 °C
-1
calibration needed -
Mass (protein) 300 cm
2
/
µ
g d
F
, n
F
, n
C
, n
S
,
λ, Λ
adsorption, binding
Mass (lipids) 150 cm
2
/
µ
g d
F
, n
F
, n
C
, n
S
,
λ, Λ
adsorption, binding
p 50 (1000) bar
-1
calibration needed pressure meter
flow rate 8000 h/ml calibration needed flow rate meter
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• Incoupling laser light for total internal reflection Incoupling laser light for total internal reflection
• Incoupling laser light for Raman spectroscopy Incoupling laser light for Raman spectroscopy
• Incoupling laser light for optical loss measurement in Incoupling laser light for optical loss measurement in
th thin films in films
• Incoupling laser light for SPR measurement Incoupling laser light for SPR measurement
• Incoupling laser light for fluorescent excitation Incoupling laser light for fluorescent excitation
• In situ monitoring and characterization of nanometer In situ monitoring and characterization of nanometer scale scale
thin layers thin layers
• Humidity and gas monitoring Humidity and gas monitoring
Non-biosensing applications
Non-biosensing applications
of
of
the optical grating-coupler waveguide sensor
the optical grating-coupler waveguide sensor
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Thank you for your kind attention
Thank you for your kind attention
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