owls presentation overview 20070402

8/15/2019 OWLS presentation Overview 20070402

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Biosensor technique based onBiosensor technique based on

Optical Waveguide LightmodeOptical Waveguide Lightmode

Spectroscopy - OWLSSpectroscopy - OWLS

20072007 MicroVacuumMicroVacuum

Copyright 200Copyright 20044, MicroVacuum Ltd. All Rights Reserved., MicroVacuum Ltd. All Rights Reserved.

Reproduction, reprint or any other form of reuse of the whole presentation documentReproduction, reprint or any other form of reuse of the whole presentation document

or any part of it including slides, text, pictures, diagrams, logos or any other content is not allowedor any part of it including slides, text, pictures, diagrams, logos or any other content is not allowed

unless with written and signed permission from MicroVacuum Ltd.unless with written and signed permission from MicroVacuum Ltd.

www.owls-sensors.comwww.owls-sensors.com


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MicroVacuumMicroVacuum 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 inMicroVacuum is involved in international and nationalinternational and national researchresearch

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.

20072007 MicroVacuumMicroVacuumwww.owls-sensors.comwww.owls-sensors.com


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ContentContent

Introduction to OWLS techniqueIntroduction to OWLS technique

ApplicationsApplications

OWLS 1OWLS 1220 instrument and OW sensors0 instrument and OW sensors

OWLS sensing theoryOWLS sensing theory

Experimental procedureExperimental procedure



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Introduction of OWLS biosensing techniqueIntroduction of OWLS biosensing technique

Optical grating coupler waveguide sensorOptical grating coupler waveguide sensor

Planar optical waveguide on glass substratePlanar optical waveguide on glass substrate Grating incorporated in planar waveguideGrating 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 moleculesSurface of the waveguide is exposed with solution containing molecules The spectrum shifts due to molecular interactions with the surfaceThe spectrum shifts due to molecular interactions with the surface

Main featuresMain features

Highly sensitive techniqueHighly sensitive technique

Real-time monitoring of surfaceReal-time monitoring of surface

interactionsinteractions

No labeling requiredNo labeling required

AAbsolute number of adsorbedbsolute number of adsorbed

moleculesmolecules cancan be determinedbe determined

Conformational change informationConformational change information

can be deducedcan be deduced



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Main Biosensing Applications of OWLSMain Biosensing Applications of OWLS

Ligand/receptor bindingLigand/receptor binding

ImmunosensImmunosensiingng (antibody/antigen(antibody/antigen))

Lipid bilayers - biomembranesLipid bilayers - biomembranes

Protein - DNA interactionsProtein - DNA interactions

Biomaterials interaction of surface with blood plasma, serumBiomaterials interaction of surface with blood plasma, serum

and proteinsand proteins

Interactions with cells toxicology, cell-surface interactionsInteractions with cells toxicology, cell-surface interactions

Monitoring environmental pollutionMonitoring environmental pollution

Analysis of association and dissociation kineticsAnalysis of association and dissociation kinetics

Kinetics of adhesion, growth and spreading of living cellsKinetics of adhesion, growth and spreading of living cells

Molecular self-assembly & nanoscienceMolecular self-assembly & nanoscience

more details in www.more details in www. owls-sensorsowls-sensors.com.com



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OWLS 1OWLS 12200Optical Waveguide Light-mode Spectroscopy SystemOptical Waveguide Light-mode Spectroscopy System



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Measuring UnitMeasuring Unit

Optical-mechanical systemOptical-mechanical system

Electronic signal processing and controlling unitElectronic signal processing and controlling unit

Industrial PC based computer withIndustrial PC based computer with

hard disk, CD readerhard disk, CD reader,,USB flash memoryUSB flash memory

Integrated sensor holderIntegrated sensor holder

Temperature control subunitTemperature control subunit

Sample injection subunitSample injection subunit

External monitor, keyboard and mouseExternal monitor, keyboard and mouseareare connected on the back panel of the Measuring Unit.connected on the back panel of the Measuring Unit.

BioSense softwareBioSense software

Microsoft WindowsMicrosoft WindowsXPXP based software provides flexible control of thebased software provides flexible control of themeasurement, data evaluation, analysis and storage.measurement, data evaluation, analysis and storage.



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OWLSOWLS Optical-mechanical systemOptical-mechanical systemschematic drawingschematic drawing

laserlaser

rotatorrotator

flowcellflowcell

photodiodephotodiode

waveguidwaveguid

eesubstratesubstrate

shuttershutter

beam mirrorbeam mirror



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Periodicity: 2400 lines/mmPeriodicity: 2400 lines/mm

Depth: 10-20 nmDepth: 10-20 nmGrating width (l) 2 mmGrating width (l) 2 mm

Waveguide layerWaveguide layer

Thickness 200 nmThickness 200 nmRefractive index 1Refractive index 1..88

Typically: SiTypically: SixxTiTi1-x1-xOO22x=0x=0..2525 00..0505

Glass substrateGlass substrate

Length 12 mmLength 12 mmWidth 8 mmWidth 8 mm

Thickness 0Thickness 0..55 mm55 mm

Refractive index 1Refractive index 1..5353

OW 2400OW 2400

optical grating coupler waveguide sensoroptical grating coupler waveguide sensor

GratingGrating


Inorganic/organic coatings are available on request.Inorganic/organic coatings are available on request.

Ir

F(wav u )S(subr )

Grating

W

L

H


Dot mark

F (waveguide layer)

S ( substrate)glass

I

W

H

Grating

L


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Inorganic coating:Inorganic coating:

SiOSiO22, TiO, TiO

22, Ta, Ta

22OO

55, ZrO, ZrO

22, Nb, Nb

22OO

55, Al, Al

22OO

33

Electrochemical/optical sensor: ITOElectrochemical/optical sensor: ITO

Organic/biofunctional coating:Organic/biofunctional coating:

Teflon Teflon

amino groupsamino groups

epoxyepoxy

carboxylatecarboxylate

biotinated surfacebiotinated surface

Surface modificationsSurface modifications



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covercover medium (C)medium (C)

glass substrate (S)glass substrate (S)

waveguide layer (F)waveguide layer (F)added layer (A)added layer (A)

nnSS

nnFF, d, dFFnnAA, d, dAA

laser lightlaser light

nnCC

Optical grating coupler waveguide sensorOptical grating coupler waveguide sensor modelmodel

EM distributionEM distribution

OWLS sensing theoryOWLS sensing theory

ZZCCAA

FF

SS

Evanescent fieldEvanescent field ZZ 200 nm200 nm

20072007MicroVacuumMicroVacuum Ltd.Ltd.www.owls-sensors.comwww.owls-sensors.com


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Basic calculation procedureBasic calculation procedure

Incoupling conditionIncoupling condition

k

)sin(nN TEairTE +

k)sin(nN TMairTM +


The effective refractiveThe effective refractive

indicesindices N(TE), N(TM)N(TE), N(TM) of theof thewaveguidewaveguide

structurestructure are calculated onare calculated on

the basis of incouplingthe basis of incoupling

condition.condition.

The incoupling anglesThe incoupling angles ((TE),TE), (TM)(TM) of electric and magneticof electric and magnetic

modes are evaluated from themodes are evaluated from the

measured lightmode spectra.measured lightmode spectra.



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Waveguiding mode conditionsWaveguiding mode conditions

+

+

+

22F

2S

22

S

F

22F

2C

22

C

F

A2

F

2

C

2

A

2

C

2C

2F

2C

2A

F22

F

Nn

nN

n

n

arctanNn

nN

n

n

arctan

d

1n

N

n

N

1n

N

n

N

nn

nndNnk0

Two equations:Two equations:

N= NN= NTETE andand =0 for TE=0 for TEN= NN= NTMTM andand =1 for TM=1 for TM

Mode equations for four-layer waveguide structureMode equations for four-layer waveguide structure

TTwowo independentindependent parameters can be calculatedparameters can be calculated



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The waveguide layer parameters nThe waveguide layer parameters nFF and dand dFF are calculated from theare calculated from the

mode equations providing that dA=0 -mode equations providing that dA=0 - three-layer model.three-layer model.

Calculation of nCalculation of nFF and dand dFF


Calculation of nCalculation of nAA , d, dAA and Mand MSensor with added layer Sensor with added layer four-layer modelfour-layer modelThe refractive indexThe refractive index (n(nAA)) and the thicknessand the thickness (d(dAA)) of the added layerof the added layer

are calculated.are calculated.

Using the model that the refractive index in the adsorbed layerUsing the model that the refractive index in the adsorbed layer

linearlylinearly depends on the concentration of the adsorbed material,depends on the concentration of the adsorbed material,the mass per unit areathe mass per unit area (M)(M) of the adsorbed materialof the adsorbed material

can be calculated.can be calculated.

dc/dn

nndM CAA

= For most protein adsorptionFor most protein adsorptiondn/dc = 0,182 cmdn/dc = 0,182 cm33/g/gwww.owls-sensors.comwww.owls-sensors.com


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OWLS spectrum Peak measurementOWLS spectrum Peak measurement

Incoupled light intensityIncoupled light intensity

versus angle of incidenceversus angle of incidence

of the laser lightof the laser light

TETETMTM

FWHMFWHM

NNTETE((TETE), N), NTMTM((TMTM))nnFF(N(NTETE, NNTMTM, n, nCC, n, nSS))ddFF(N(NTETE, NNTMTM, n, nCC, n, nSS))nnAA(N(NTETE, NNTMTM, n, nCC, n, nSS, n, nFF , d, dFF))ddAA(N(NTETE, NNTMTM, n, nCC, n, nSS, n, nFF , d, dFF))M (nM (nAA,, ddAA, dn/dc), dn/dc)



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Monitoring surface processes RIM measurementMonitoring surface processes RIM measurement Repeating the Optical Waveguide Light-mode Spectrum measurementsRepeating the Optical Waveguide Light-mode Spectrum measurements

Time-dependence of the optical parameters are presented on graphTime-dependence of the optical parameters are presented on graph

and analyzed.and analyzed.

..

NNTETE(t), N(t), NTMTM(t),(t),

nnAA(t), d(t), dAA(t), M(t)(t), M(t)


..


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A typical example of an OWLS experiment using proteinsA 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)

Absorbedmassofprotei

(ng/cm2)

BaselineBaselinein bufferin buffer

Shift of theincoupling angles

00..4mg/ml4mg/ml

BSABSA

bufferbuffer

Baseline-runBaseline-runin pure (non-filmin pure (non-film

forming) buffer : determinationforming) buffer : determination

of the refractive indexof the refractive index nnFF

and theand the

thickness,thickness, ddFF, of the waveguide ., of the waveguide .

Eliminates small differences inEliminates small differences in

sensor parameters from onesensor parameters from onechip to other.chip to other.

Adsorption experimentAdsorption experiment monitorsmonitors

the evolutionthe evolution ooffnnAA,, dd

AAand M. Inand M. In

situ determination of adsorbedsitu determination of adsorbed

mass and the kinetics of adlayermass and the kinetics of adlayer

formation.formation.

Desorption phase:Desorption phase: this is athis is awashing stepwashing step iin pure buffer,n pure buffer,

which provides informationwhich provides information

about the stability of theabout the stability of the

formed adlayer and possibleformed adlayer and possible

desorptiondesorption kinetics.kinetics.



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0

100

200

300

400

500

600

0 20 40 60 80 100 120 140

Time (min)

Mass(ng/cm2)

GA

TRIS

MoAb

HCl

Blocking

HCl

Sensitization of the waveguide sensors with monoclonal anti-HSP70 antibodiesSensitization of the waveguide sensors with monoclonal anti-HSP70 antibodies

GAGA -- injection of glutaraldehydeinjection of glutaraldehyde

(2.5% in distilled water) to activate(2.5% in distilled water) to activate

amino groups,amino groups,

TRISTRIS - changing distilled water to- changing distilled water totris buffer (42mM, pH 7.4),tris buffer (42mM, pH 7.4),

MoAbMoAb - injection of monoclonal- injection of monoclonal

anti-HSP70 IgG solution (33anti-HSP70 IgG solution (33g/mlg/mlin tris buffer) to form the sensitivein tris buffer) to form the sensitive

layer,layer,

HClHCl - injection of hydrochloric acid- injection of hydrochloric acid

(10mM) to wash the surface,(10mM) to wash the surface,

BlockingBlocking - blocking free reactive- blocking free reactive

aldehyde groups with glycinealdehyde groups with glycine

(10mM in tris buffer)(10mM in tris buffer)

The experiment was performed with a FIA system with injection loop volume of 200 l and with flow rate of 90 l/minwww.owls-sensors.comwww.owls-sensors.com


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HSP70 protein binding to immobilizedHSP70 protein binding to immobilized anti-HSP70 antibodiesanti-HSP70 antibodies

The lowest detectable amount of HSP70

is 1 pg/ml.


250

300

350

400

450

500

100 150 200 250 300

Time (min)

Sensorresponse(a.u.)xx

1 pg/ml

5 pg/ml10 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


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Model experiment performed on amino functionaliModel experiment performed on amino functionalizzed surfaceed surface


0 20 40 60 80 100 120

Time (min)

0

100

200

300

400

500

600Coverage (ng/cm2)

1 2 34

5

A

BC

D

r

r

rrr

r

A - distilled water

B - 2.5% glutaraldehydeC - TRIS buffer, pH=7.4

D - 200 g/ml BSA (TRIS)

r - regeneration solution, 0.1 M HCl

1 - 10g/ml IgG

2 - 25g/ml IgG3 - 50g/ml IgG

4 - 100g/ml IgG

5 - 200g/ml IgG

BSA

anti-BSA IgG



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ParametersParameters thatthat OWLS canOWLS can mmeasureeasure

Parameter Sensitivity (*10-6

) Known parameters Possible use

nF 700 000 nC, nS, , gas detection, swelling

dF 860 nm-1

nC, nS, , gas detection, swelling

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

dA 210 nm-1

dF, nF, nC, nS, , SAM, protein size

pH 50 calibration needed pH meter

T 10 C-1

calibration needed -

Mass (protein) 300 cm

2

/g dF, nF, nC, nS,

, adsorption, binding

Mass (lipids) 150 cm2/g dF, nF, nC, nS, , adsorption, binding

p 50 (1000) bar-1

calibration needed pressure meterflow rate 8000 h/ml calibration needed flow rate meter



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Incoupling laser light for total internal reflectionIncoupling laser light for total internal reflection

Incoupling laser light for Raman spectroscopyIncoupling laser light for Raman spectroscopy Incoupling laser light for optical loss measurement inIncoupling laser light for optical loss measurement in

ththin filmsin films

Incoupling laser light for SPR measurementIncoupling laser light for SPR measurement

Incoupling laser light for fluorescent excitationIncoupling laser light for fluorescent excitation

In situ monitoring and characterization of nanometerIn situ monitoring and characterization of nanometer scalescale

thin layersthin layers

Humidity and gas monitoringHumidity and gas monitoring

Non-biosensing applicationsNon-biosensing applications ofofthe optical grating-coupler waveguide sensorthe optical grating-coupler waveguide sensor

20072007 MicroVacuumMicroVacuum Ltd.Ltd.www.owls-sensors.comwww.owls-sensors.com


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Thank you for your kind attentionThank you for your kind attention


owls presentation overview 20070402

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