surface plasmon resonance (spr)...

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