Surface Plasmon Resonance

BOC tutorial 12/10/2015

Once upon a time…

Wood, R W. 1902. Philosophical Magazine 4: 269–275.

anglegrating

silver

Wavelength (Å)

Angle

of

Inci

den

ce

Rayleigh, L. 1907. “On the Dynamical Theory of Gratings.” Proceedings of the Royal Society A 79 (532) (August 2): 399–416.

A few years later: Gratings explained

Surface Plasmons:electron oscillations on a dielectric interface

Can be excited by coupling to a photon with the same wave vector: ‘disappearing photons’

Excited surface plasmons propagate like a wave over the surface until the energy is absorbed or emitted

Surface Plasmons

Wavefunction of plasmons on metal-dielectric (ksp) interface depends on wavelength and dielectric constants

Wavefunction of light parallel to the metal surface (kx) depends on wavelength, dielectric constant and angle of incidence

In a thin metal film, the light wave on the surface can affect the plasmon wave on the other side

At some angle θ, kx will equal ksp ie. the two waves are equal and the plasmon wave will start to resonate

Resonance of Surface Plasmons

θ

Liedberg, B., C. Nylander, I. Lundstrom. 1983. Sensors and Actuators 4: 299–304. Kretschmann, E, and H Raether. 1968. Zeitschrift Für Naturforschung 23a: 2135–2136.

Resonance is observed as a narrow minimum in intensity of reflected light

Changes to the refractive index on the metal-dielectric interface change the dielectric constant of the interface (like different medium or a layer of molecules)

If the dielectric constant changes, the wavefunction changes and thus the angle where resonance occurs

Useful as a measuring instrument

Liedberg, B., C. Nylander, I. Lundstrom. 1983. Sensors and Actuators 4: 299–304.

Real-time Label free Quantitative analysis

Now also multiplex

KD in nM to mM range Needs heavy molecules as analyte Assay in pure or non-pure samples

SPR measurement

Saturation measurement Similar to other techniques (ITC, ELISA)

Equilibrium constants

[AB]

Fit a binding curve

Dynamic constants

Finding the proper conditions◦ Immobilization◦ Buffers, flow rate◦ Regeneration

Non-specific binding & Referencing

Surface effects

Modelling: Does the model describe the experiment?

Pitfalls

Anti-citrullinated protein antibody from RA patients

Specific glycosylation in Fab◦ Highly present in some patients

Effects on affinity?

Compare glycosylated and non-glycosylated variant

A simple example

Initially: it does not fit

Two phases visible in sensorgram

Likely cause: Mass transport limitation

Fitting I

2000 4000 6000 8000-1000

0

1000

2000

3000

4000

50002001005025

Time [s]

Res

po

nse

[R

U]

Fitting II

Wild-type antibody Non-glycosylated antibody

KD = 60 nM KD = 10 nM

Results

E4wt

E4NG

0.01

0.1

1

10

100

3X

4X

5X

7X

8X

11X13X15X

16X

17X

18X19X

KD [

nM

]

F3wt

F3NG

1

10

100

2X3X

4X

6X7X

8X

11X

15X16X

18X20X

2X3X4X6X7X8X11X15X16X18X20X

F3wt40.060.05.515.023.06.611.07.99.429.036.0

F3NG8.811.03.95.16.52.14.33.23.16.56.2

KD [

nM

]