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
]