8/8/2019 Optical Spec 7 - Surface Plasmon Resonance

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Physical Biochemistry Surface Plasmon Resonance

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A technique to study binding of analytes to ligands

The analyte flows through the flow cell (F)

Immobilised ligand coats substrate (S) which is attached to a prism (P)

The light source (L) illuminates the substrate with a thin wedge of light

TIR occurs at the surface of the substrate

A detector array (D) records the reflected wedge of light

The detector is made up of small cells arranged along a line

Each detector will detect light being reflected at a certain angle

At most angles there would be constant reflection, but at certain

angles the amount of reflected light would fall, due to surface

plasmon resonance.When a ligand is bound to the substrate, the SPR dip will occur at a certain angle, when analyte molecules

bind to the ligand, the dip will sh ift. We measure how this dip shifts with angle as a function of time.

The Sensor Chip:

A 3D matrix has the ligand immobilised onto it. This increases the density of the ligand / surface area.

Applications:

The change in SPR is directly proportiona l to mass being immobilised Useful for equilibrium and kinetic measurements

No labelling required but has to be immobilised

Advantages: No labelling Real time (fast measurements, sub-second time scale)

8/8/2019 Optical Spec 7 - Surface Plasmon Resonance

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Physical Biochemistry Surface Plasmon Resonance

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Can use very small amounts of sample volume (180Da, reliable measurements with masses >1000Da

What is a plasmon? A plasma is a medium with an equal concentration of + and charges of which at least one charge

species is mobile. M etal can be regarded as a plasma (e - can move) Plasma has a natural oscillation frequency (plasma frequency) where the plasma oscillates as one.

The plasma can oscillate at other frequencies. Plasmons = plasma oscillations Surface plasmon is a plasma oscillating along the surface of the metal The plasma frequency of the surface plasmon is very sensitive to the properties of the surface. Ligand

attachment and subsequent analyte attachment will change the surface plasmon frequency.

The plasmon is probed using TIR

How is energy coupled from the photon to the plasmon? The evanescent waves from TIR propagate slightly into the surrounding medium (gold) The angle of the TIR will influence the wavelength of the evanescent wave The frequency, wavelength and polarisation of E M R needs to be matched to the plasmon When these are matched to the plasmon, energy from light can be transferred to the plasmon. This

only occurs at a certain angle as only at certain angles will the wavelength of the evanescent wave

match that of the plasmon. This is seen as a dip. Binding at the surface causes a change in the refractive index for the evanescent wave