Molecular interactions observed in a real time

Bogdan WalkowiakDepartment of Biophysics

Technical University of Lodz

and

Department of Molecular and Medical Biophysics, Medical University of Lodz

– possibilities and limitations of

SPR technology

Endpoint assay

Introducing a binding profile

TIR – Total Internal Reflection

SPR – Surface Plasmon Resonanse

kx=2n1sin(/

ksp=(ng2n2

2/ng2+n2

2)0.5

SPR Detection System

Sensor chip withgold film

Prism

Flow channel

Light source

Polarizedlight

Optical detection

unit

Reflected light

III

I II

II

I

Sensorgram

Microfluidic system IFC

Liquid Handling

Gold-Dextran Surfaces

• Biocompatible

• Low non-specificbinding

• More than 100 runs on the same surface

Specific layer

Dextran layer

Linker layer

Gold film

Glass

12

14

16

The Sensorgram

ResonanceSignal (KRU)

Dissociation

Regeneration

Ass

oci

atio

n Kinetics

Concentration

100 200 300 400 500 600

Time (s)

18

BiaCore sensor

BiaCore sensor family

Correlation between SPR response and surface concentration

Signal proportional to massSame specific response for different proteins

-50

0

50

100

150

200

0 50 100 150 200 250 300 350 400 450 500

time (s)

resp

onse

(R.U

.)

pH 6.8

pH 4.0

pH 2.8

Protein concentration at the sensor surface

Coupling Chemistries

Protein immobilization at the sensor surface

16750

21750

26750

31750

36750

41750

0 200 400 600 800 1000 1200 1400 1600 1800

time (s)

resp

on

se (

R.U

.)

activation

concentration andimmobilization

saturation

-200

0

200

400

600

800

1000

1200

0 50 100 150 200 250

time (s)

resp

on

se (R

.U.)

fibrin, Sta K2, 25 ug/ml

fibrin, Staphylo, 25 ug/ml

dextran, Sta K2, 25 ug/ml

dextran, Staphylo, 25 ug/ml

Szemraj J., Pietrucha T., Nowak P., Walkowiak B. In preparation 2002

Recombinant protein mediating fibrynolyitc process

0

50

100

150

200

250

300

0 50 100 150 200 250 300 350 400

time (s)

resp

onse

(R.U

.)

pH 8,2

pH 8,4

pH 8,0

pH-dependence of MBP and DNAK interaction

Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

0

50

100

150

200

250

300

7,2 7,4 7,6 7,8 8 8,2 8,4 8,6 8,8

pH

resp

onse

(R.U

.)

Final presentation of pH-dependence for MBP and DNAK

Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

0

200

400

600

800

1000

1200

1400

0 50 100 150 200 250 300 350 400 450 500

time (s)

resp

onse

(R.U

.)

specif ic antibody binding to MBP protein

nonspecif ic binding

Specific antibody binding to immobilized MBP protein

Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

-20

0

20

40

60

80

100

0 50 100 150 200 250 300 350 400

time (s)

resp

onse

(R.U

.)

DNAK protein binding to MBP proteinDNAK protein binding to MBP protein in the presence of specific antibody

Specific binding inhibition by Mab against MBP protein

Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

-50

0

50

100

150

200

250

300

350

400

0 50 100 150 200 250 300

time (s)

resp

onse

(R.U

.) 12.5 uM 25.0 uM 50.0 uM100.0 uM

Acid glycoprotein binding to immobilized PAI-1

Boncela J., Papiweska I., Fijałkowska I., Walkowiak B., Cierniewski CS. J..Biol.Chem,276, 35305, 2001

-100

0

100

200

300

400

500

600

0 50 100 150 200 250 300 350

time (s)

resp

onse

(R.U

.)8 nM16 nM32 nM64 nM

Vitronectin binding to immobilized PAI-1

Boncela J., Papiweska I., Fijałkowska I., Walkowiak B., Cierniewski CS. J..Biol.Chem,276, 35305, 2001

Glass plate one side covered with gold film

Carbon deposition by RF CVPD technology

Preparation of sensor surface for NCD-plasma proteins interaction

Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

500

1000

1500

2000

2500

3000

3500

4000

500 1000 1500 2000 2500 3000 3500

R (1/cm)

Ram

an

sp

ectr

a in

ten

sit

y (

a.u

.)NCD surface

gold surface

Raman spectrometry analysis of prepared sensor

surface

Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

-200

0

200

400

600

800

1000

1200

1400

0 100 200 300 400 500 600 700 800 900 1000

observation time (s)

mas

s ch

ange

on

the

sens

or s

urfa

ce (R

.U.)

gold surface

NCD surface

platelet poor plasma rabbit serum anti-fg

plasma proteins adsorbed on the sensor surface

antibodies bound to fibrinogen

Adsorbtion of plasma proteins to the NCD surface

Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

Sensor for detection of platelet adhesion

to immobilized fibrinogen

Immobilization of rabbit anti-fibrinogen at the sensor surface

Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

-200

-100

0

100

200

300

400

500

0 200 400 600 800 1000 1200 1400

observation time (s)

mas

s ch

ang

e o

n t

he

sen

sor

surf

ace

(R.U

.)

fibrinogen suspension of blood platelets

bound fibrinogen

adheredplatelets

regeneration

Adhesion of blood platelets to the sensor with immobilized fibrinogen

Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

Cells interaction with immobilized ligands

0

1000

2000

3000

4000

5000

6000

7000

0 10 20 30 40 50 60 70 80 90 100

time (s)

resp

onse

(R.U

.)140 mM NaCl

250 mM NaCl

500 mM NaCl

Effect of high salt concentration on antibody-ligand interaction

Effect of high salt concentration on antibody-ligand interaction

-10

40

90

140

190

240

290

340

390

0 10 20 30 40 50 60 70 80 90 100

time (s)

resp

onse

(R.U

.)140 mM NaCl

250 mM NaCl

500 mM NaCl

Sample Recovery and Fractionation

563

Fractionate dissociating analyte for- re-injection

- sequencing

- mass spectrometry

- electrophoresis

- PCR

Recover eluted material for- enzymatic transfer assays

- on-surface cleavage processes

- alterations due to binding or regeneration

Recover excess sample during injection

- save expensive sample and reagents

BiaCore - MS

Limitations

Molecular weight of analyte not less than 200

Limitet chemical resistivity to organic solvents

Residual interaction of analyte with dextran matrix

Active for measuremet distance from a sensorsurface is limited to 300 nm

Thank you very much

for your attention