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Surface Plasmon Resonance (SPR)
SpectroscopyTheory, Instrumentation & Applications
Antonella Badia
antonella.badia@umontreal.ca
CHEM 634
McGill University
January 26, 2007
Faculté des arts et des sciencesDépartement de chimie
2
SPR Spectroscopy - Overview:
The detection principle relies on an electron chargedensity wave phenomenon that arises at the surfaceof a metallic film when light is reflected at the film underspecific conditions.
Molecular adsorption/desorption events are measured asa change in the refractive index at the metal film surface(“sensing surface”).
Advantages:– Label-free detection technique– Distinguishes surface-bound material from bulk material– Monitor molecular interactions in real-time (kinetics)– Highly-sensitive ( dfilm of ~1-2 Å or nanograms of adsorbed mass)– Works in turbid or opaque samples
3
Biological Applications of SPR
Protein/Protein
RNA/DNAProtein/DNA
Protein/Carbohydrates
NH
OH
NH
NH
NH2
NH2
NH
O
O
ONH
OHOH
O
O
Protein/Peptide
DNA/DNA
4
Basic Principle
• A binding molecule is bound to the sensor surface(eg. a peptide)
• Another (the analyte) is passed over the surfaceand binds to it.
5
Other Areas of Application
Self AssembledMonolayers (SAM)
Electrochemistry
S
Fe
S
Fe
- e-
+ e-
S
Fe
S
Fe
S
Fe
S
Fe
S
Fe
S
Fe
Interfacial Reactions
S
R
A
B
S
R
A
B
S
R
A
B
S
R
A
B
S
R
A
B
S
R
A
B
S
R
A
B
S
R
A
B
MUA
- - - - - - - - - -
PEI
++
++
++
+
+++
+ ++
+++
+ ++
+++
+ ++- - - - - - - - - -
HA
--
--
--
--
-
+++
+ ++
+++
+ ++
+++
+ ++- - - - - - - - - -
- ---
-
-
-- - -
-
-
--
++
+
++
CH
+++
+ ++
+++
+ ++
+++
+ ++- - - - - - - - - -
- ---
-
-
-- - -
-
-
--
+++
+
+
++
+
+
+
+ ++
+
+
+++
+
+
-
-
- ---
HA,C
H,etc
...
PE Multilayers
6
Surface plasmons
Plasmons are collective charge density oscillations of the nearlyfree electron gas in a metal.
Plasmons can be excited both in the bulk and on the surface ofa metal.
Surface plasmons or surface plasmon polaritrons are surfaceelectromagnetic waves that propagate parallel along ametal/dielectric interface.
Surface plasmons
7
Existence of electronic surface plasmons
Conditions are met in the IR-visible wavelength region for air/metaland water/metal interfaces (where m is negative and d of air orwater is positive). Typical metals that support surface plasmons aresilver and gold.
Electronic surface plasmons obey the following dispersion relation:
real metal ( m)
real dielectric ( d)
Conditions to be met: real of the metal must be negative | real| of metal > real of dielectric
kSP =
cd m
d + m
8
SPR vs. LSPR
The excitation of surface plasmons by light is denoted as:
surface plasmon resonance (SPR) for planar surfaces
localized surface plasmon resonance (LSPR) for nanometer- sized metallic structures.
Surface plasmon polaritron Localized surface plasmon
9
Total Internal Reflection (TIR) phenomenon
i
n2 < n1TIR for
non-absorbing media
The fully reflected beam leaks an electrical field intensity (i.e. evanescent fieldwave) into the low refractive index medium.
No photons exit the reflecting surface but their electric field decreasesexponentially with distance from the interface, decaying over a distanceof ~1/4 wavelength beyond the surface.
If the lower refractive index media has a non-zero absorption coefficient, theevanescent field wave may transfer the matching photon energy to themedium.
Refle
ctiv
ity (
a.u
.)
c
10
SPR phenomenon
Under specific conditions (i.e. incident angle of the light beam or wavelength ),the electromagnetic field component of the p-polarized light penetrates themetal layer, and energy is transferred to the metal’s electrons.
This energy transfer produces surface plasmon polaritrons at the metal-mediuminterface.
As a result of the energy transfer, there is a decrease in the reflected lightintensity (gray region) at a specific angle of incidence.
TIR-interface coated with a thin metal layer
11
• The surface plasmon wavepropagates in the x- and y-directions along the metal-dielectric interface, for distancesof ~ tens to hundreds of micronsand decays evanescently in thez-direction (into the low refractiveindex medium) with 1/e decaylengths on the order of 200 nm.
• Due to its electromagnetic andsurface propagating nature, thesurface plasmon wave enhancesthe evanescent electricfield amplitude.
y
(SPR-evanescent wave)
Au film (SPR-evanescent wave)
no Au film (non-absorbing TIR)
SPR-evanescent wave
12
Characteristics of the SPR evanescent wave
13
Surface plasmon excitation: energy and momentum
matching
• For plasmon excitation by a photonto take place, the energy andmomentum of these quantum-particles must both be conservedduring the photon transformationinto a plasmon.
• This requirement is met when thewavevector for the photon kph andplasmon ksp are equal in magnitudeand direction for the samefrequency of the waves.
• Light falling directly on the metal-dielectric interface cannot coupleinto the surface plasmon sincematching of both and kx is notpossible.
• For coupling to take place, thevalue of kx of the incident lightmust be increased.
r k ph <
r k sp (for all )
ksp =c
m d
m + d( )
kph =
cd
14
Surface plasmon excitation: prism couplers
at the resonance angle o
photon
High-indexprism coupler
p > d
15
Because the plasmon electric field penetrates a short distanceinto the lower refractive index medium, the conditions for SPRare sensitive to the refractive index n at the metal-dielectricinterface.
A change in the bulk refractive index of the dielectric mediumand the adsorption or desorption of molecules from the metalsurface changes the refractive index at the metal-dielectricinterface and results in a change in the velocity of the plasmons.
This change in plasmon velocity alters the incident light vectorrequired for SPR and minimum reflection.
The exact position of resonance bears information on theinterfacial mass coverage/thickness of the interfacial layer.
Effect of molecular adsorption/binding on SPR
16
SPP excitation configurations
Otto configuration
Kretschmann ATR configuration
(thin gap)
17
SPR-based measurements
• Resonance angle shift
• Imaging/microscopy
• Wavelength shift (FT-SPR)
The aim of SPR instrumentation is to determine the
resonance position as precisely as possible and with the
best time resolution.
18
SPR-based measurements
• Resonance angle shift
• Imaging/microscopy
• Wavelength shift (FT-SPR)
19
Resonance angle shift measurements: principleR
efle
ctiv
ity (
a.u
.)
c o
• Metal-coated high-refractive indexprism (BK7, sapphire, LaSFN9,SF10, etc.)
• ATR/Kretschmann configuration
• Single wavelength p-polarizedincident light
• The reflected light intensity ismeasured as a function of the angleof incidence .
• The angle scan changes the wave-vector kx of the incident light ontothe prism base.
20
Angle-shift design 1
• A lens is used to focus the lightbeam onto the prism base.
• Within the focus, a variety ofangles of incidence are covered.
• The angle range (typically a fewdegrees) is given by the focallength of the lens and the beamdiameter.
• The reflection curve is monitoredby a PSD or a linear CCD array.
• An array scan of reflectivity vs.pixel number is obtained whichcannot be modeled usingFresnel equations.
Commercial instruments:Biacore, Reichert SR7000
21
Angle-shift design 2
• The laser and detector armare moved synchronouslyusing a -2 goniometerand the reflected lightintensity is measured as afunction of the angle ofincidence.
• The resulting reflectivity vs.incidence angle plot can bemodeled using Fresnelequations.
Commercial instruments:Biosuplar, Resonant Probes,Optrel Multiskop
22
SPR angular reflectivity curve
ATR angle
o
Width1/2
23
Surface interactions
– Direct coupling of ligand (binding molecule) to surface
– Membrane anchoring, where the interacting ligand is on the surfaceof a captured liposome
– Indirect, via a capture molecule (eg. a specific IgG)
24
Fabrication of sensing surface
Coat glass slides or prism with 45-50 nm of gold
Surface Chemistry
Hydrophilic and hydrophobic surfaces
Immobilize ligand
Direct coupling - attach ligand chemically via a linker
Capturing - attach a protein that binds your target
References: Jonsen et. al 1991 (Anal Biochem 198, 268-277);O’Shannessy et. al., 1992 (Biochem 205, 132-136)
25
Allows covalent coupling via -NH2, -SH, -CHO & -COOH groups:
Ligand coupling chemistry
26
adsorbate solution
detectorlaser
Au filmadsorbate layer
Molecular adsorption and
angle shift
detectorlaser
Au filmH2O
H2O
detectorlaser
Au filmadsorbate layer
molecular adsorption
rinse with H2O
27
Monitoring molecular adsorption by SPR
adsorbate injection
II
I
time
Injection of
adsorbate
1
2
t
28
The The sensorgramsensorgram
o (°)
( o d)
29
Sensorgrams for reversible and
irreversible adsorption
o (
°)
30
Analysis of SPR sensorgram
• How much? Active surface or bulk concentration
• How fast? Kinetics
• How strong? Affinity
• How specific? Specificity
31
Surface concentration
• Resonance angle changeis proportional to masschange (mass of boundmaterial).
• The change in surfacerefractive index isessentially the same for agiven mass concentrationchange (allowsmass/concentrationdeductions to be made).
• Example: same specificresponse for differentproteins
32
Adsorbate film thickness calculated as an
average value
(°)
% R
efle
ctiv
ity
o(°)
Bare Au
+ Adsorbate
adsorbatelayer
o = c1 n + c2 d
d d
33
Modeling with Fresnel equations:
Winspall software (freeware, Wolfgang Knoll, MPI-P)
Prism
o = c1 n + c2 d
34
Glass Slide
Modeling with Fresnel equations: Winspall
Prism
35
Modeling with Fresnel equations: Winspall
Prism
Glass Slide
Ti
Au
36
Modeling with Fresnel equations: Winspall
Prism
Glass Slide
Ti
Au
Thiol
S
Fe
S
Fe
S
Fe
S
Fe
37
Modeling with Fresnel equations: Winspall
Prism
Glass Slide
Ti
Au
Thiol
S
Fe
S
Fe
S
Fe
S
Fe
Water n2 < n1
n2
n1
38 (°)
% R
efl
ec
tiv
ity
SPR profile fit
Fit
Experimental
39
Calculation of surface concentration
Determine adsorbate film thickness (dfilm) from Fresnel fitting ofthe experimental angular reflectivity curve
Determine the incremental change in the bulk refractive indexwith concentration of the adsorbate ( nadsorbate/ c) usingrefractometry
The surface excess ( / mol·cm-2) is calculated according:
= d(nfilm nsolvent )
1
nadsorbate / c
n of hydrocarbon films 1.45-1.50 (589-633 nm)
40
Binding kinetics
Time
Reso
nance
Req
B
A
B
1 2 3
1
2
3A + B AB kon
AB A + B koff
41
Simple binding kinetics
A + B AB K =
kon
koff
d[AB]
dt= kon [A][B] koff [AB]
• If flow rate of A is sufficiently high, [A] = a0
• We can also write [B] = b0 - [AB]
• SPR signal [AB]
• Rmax b0 (measured if all B bound to A)
equilibrium constant forAB complex formation
42
• We may write:
• At equilibrium R = Req and dR/dt = 0.
• It follows that:
• The value of K can be obtained from measurements of Req for a series of a0
dR
dt= kona0(Rmax R) koff R = kona0Rmax (kona0 + koff )R
Req = Rmax
a0K
a0K +1
43
Mass transport considerations
• Do mass transport limitations impact the rate constants?
– Yes, if binding rate > diffusion rate
– Introduces gradients
– Myszka DG, et. al., “Extending the range of rate constantsavailable from BIACORE: interpreting mass transport-influenced binding data”, Biophys J 1998, 75: 583-594.
44
SPR-based measurements
• Resonance angle shift
• Imaging/microscopy
• Wavelength shift (FT-SPR)
45
SPR imaging apparatus
46
SPR Imaging
(°)
% R
efl
ec
tiv
ity
%R
In SPR “imaging”, the reflectivity change, %R, is determined by measuring theSPR signal at a fixed angle of incidence before and after selective molecularadsorption across a fixed surface.
47
SPR Imaging - Image processing
Z-resolution 1-2 ÅX-Y resolution microns
48
Problems of SPR
• Limited to choice of metal which results in SPR
• Sample preparation– Attaching probe to metal surface can prove difficult
• Non-specific interactions– Good news- Everything has an SPR signal!– Bad news- Everything has an SPR signal!
• Refractive index is temperature dependent
49
Future of SPR
• Combination of SPR with various surface analyticaltechniques:
– Electrochemistry
– Quartz Crystal Microbalance (QCM)
– Ellipsometry
– Scanning Probe Microscopy
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