OutlineOutline1. Optical methods2. Surface Plasmon Resonance (SPR)

光學生物晶片光學生物晶片

授課老師 : 林啟萬 台大醫工所副教授cwlin@cbme.mc.ntu.edu.tw

II. Optical Method

Optical methodsLight can be defined as the electromagnetic spectrum in the frequency range of 1011 (far infared) to 1017 (far ultraviolet). The energy of a single photon is given by, E=hvin J or eV, where h is Planck’s constant=6.6261x10-34 J．s = 4.1361x10-14 eV．S and υ is the frequency of light in Hz, and the wavelength of the light, λ, is given as , where c is the speed of light in vacuum=2.99792458x108 m/s.

1. UV-VIS absorption2. Fluorescence and phosphorescence

emission3. Bioluminescence4. Chemiluminescence5. Internal reflection spectroscopy6. Laser light scattering

Terms• Quantum efficiency (Q): defined as the number of carriers generated per

incident photon.• Gain: defined as the ratio of the total current that flows in response to photo

excitation to the current that flows in direct response to impinging photons (the primary photocurrent). This is a measure of the carrier multiplication or other mechanisms that go on in the sensor.

• Bandwidth or frequency response: the frequency range over which the photo detector responds, with a cut-off frequency at which the output signal amplitude is reduced by 3dB.

• Responsivity: relating to the output signal amplitude to input power, defined as

• Noise equivalent power (NEP): defined as the amount of light required to get a signal equivalent in power to that of the noise (S/N=1). For a current-output transducer:

• Detectivity (D*): defined as the reciprocal NEP, correcting for the proportionality of . in

Absorption

• Beer Lambert’s Law A = εCL

Photo-Luminance

The First observation of luminescence 1565 The first paper on luminescence 1852 (Sir G.G. Stokes)Luminance is a general term used to describe the emission phenomenon, which caused by energy absorption then followed by a radiation emission with lower energy. Depending on the type of feeding energy, there are many kinds of luminescence can be distinguished: bio-, cathodo-, chemo-, electro-, photo-, radio-, thermo-, luminescence

Mean lifetime of molecules in the excited state

10-2 secRotational transition

10-3 secVibrational transition

10-6 secElectronic transition (T – S)

10-9 secElectronic transition (S – S)

10-15 secPhoton absorption

Fluorescent process

Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence.

Competitive Fluorescent

Chemilumescence

Optical Fiber

Flow through

Attenuated Total Reflection

Laser scattering

Laser Doppler flow meter

III. SPR

1. Introduction• Solid state electronic properties can be studied by using two different

approximation:– Electrons moving in the periodic array of atoms, or– High density of free electron liquid in a metal (~1023 cm-3), ignoring the

lattice. (plasma concept)• It thus allow the longitudinal density fluctuation, plasma oscillations,

propagate through the volume of metal.• The quanta energy of these “volume plasmons” is in the order of 10 eV.

( ), which has been studied in detail theoretically and experimentally with electron-loss spectroscopy.

• Maxwell’s theory shows that SP can propagate along a metallic surface with a broad spectrum of eigen frequencies for , which depends on the wave vector k.

02 /4 mnep πω hh =

2/0 pωω L=

I. Introduction• SPs represent electromagnetic surface waves that have their intensity maximum in

the surface and exponentially decaying fields perpendicular to the surface.• They can be produced by electrons or by light in the attenuated total reflection

(ATR) device.• With the excitation by light, a strong enhancement of the electromagnetic field in

the surface (resonance amplification) can emit up to 100 times stronger in the resonance than out of resonance. This enhancement is correlated with a strong reduction of the reflected intensity up to a complete transformation of the incoming light into SP. It thus provides an important tool for the studies of metal optics on smooth and corrugated surfaces. The measurement of its intensity and its angular distribution allows determination of the surface roughness, r.m.s. height and correlation length. On structured surface, the angular distribution of diffusely scatter light can be changed engineeringly.

• The applications include– Enhanced photoeffect– Localized plasomons effect results in large field enhancement (104-106) in Nonlinear

second harmonic generation (SHG) and Surface enhanced Raman Scattering (SERS)– Scatter light amplification at Rayleigh waves– Light emission from tunnel junction– High frequency mode with ultra thin film

Surface Plasmon Resonance

SPR Principle - General

• “Surface plasmon resonance spectroscopy as a tool for investigating the biochemical and biophysical properties of membrane protein systems. I: Theoretical principle”, BBA, 1331 (1997) 117-129

– Excellent review by Z. Salamon, H.A. Macleod, G. Tollin, • Observed by Woods Lamp on metal grating early in the 20th century.• “Surface Plasmon” appears in 1960s to explain the existence of such a

phenomena.• Definitions of Surface Plasmon

– A quantized oscillation of an electron on a planar surface of a metallic film– charge density waves propagating along a metal-dielectric interface

• It can be excited by various forms of energy, e.g. optical, electrical, chemical.– Surface plasmon is excited by a resonant interaction, momentum match condition

(Ksp=Kx) with an evanescent field. Ksp: wave vector of surface plasmon, Kx : parallel component of photon wavevector

• Extensively used to study the changes in refractive index of thin film (metal, dielectric) and its vicinity surface properties (nm – sub-um) in physics and recently in biochemistry and biomedicine.

SPR Principle - General• Four basic elements

1. Light source (polarization, beam geometry wavelength, angle, intensity, and phase modulation)

2. A prism (couple photons to plasmons)3. A thin film of metal (Au, Ag, Cu, Al, Pd,

Pt, Ni, Co, Cr, W) or semiconductor (SiO2) (~ 50 nm)

4. A light detector• Two basic configurations

Kretschmann type (often used)– Otto type (air or dielectric gap)

• Three features in the responsive curve• The (angular or wavelength) position• The (angular or wavelength) width• The depth of the resonance

入射光角度調變或波長調變

石英介質 n1

金屬介質　(金銀鉑二氧化矽等) n2

生物分子固定層 n3

反射光強度變化

x

TM Polarizer

PMMA n1

金屬介質　(金銀鉑二氧化矽等) n2

生物分子固定層 n3

入射光角度調變或波長調變

反射光強度變化

S

x

p

5-100 nm

450 nm

Polarizer

SPR Principle - General• Propagation length of the SP

–In z direction intensity decrease to 1/e, L=1/kz, (@600 nm, air:280 nm, Au:31 nm)–In x direction intensity decrease to 1/e, (@ 515 nm, ~22um)

• Material dielectric constant–Gold -72+i2–Silver -81+i5–Copper -72+i7–Aluminum -173+i32

• For SPR signal, Ag is larger than Au (~4 times)

Types of SPR

• Time-resolved SPR• Steady-state SPR• Fiber SPR• SPR Imaging

Biomolecules Immobilization– Monolayer transfer onto metal film (LB film, lipid

vesicles)– Assembly techniques

• surface chemical modification: alkylsilane on hydroxylated surface (SiO2, Al2O3); alkanethiolates on Au, Ag, and Cu; alcohols and amines on Pt; Carboxylic acid on Al and Silver oxide; dextran hydrogel(Pharmacia, BIACore)

• self-assembly lipid bilayer

SPR Principle - General

• Light propagating in a dielectric medium induces polarization in dielectric medium.

• The total energy & momentum transport in the medium in the form of a coupled mode of electromagnetic field with matter.

Electromagnetic Interaction in a Dielectric System

SPR Principle - General

00

1εµ

=c

µε1

=v00εµ

µε=n

ncv =

tBE∂∂

−=×∇0=⋅∇ E

0=⋅∇ B1.2.

3.

4.tEB∂∂

=×∇ µε

εµ

:permittivity:permeability

Light Propagation velocity (wave function) in medium

Assume no free charge or free current

n: refractive index

02 =+∇ EkE c )(21

21

εεεεω+

=c

kx "' xxx ikkk +=

Light (EM Wave) in a linear homogeneous mediumGoverned by Maxwell Equation

Theory of SPR – Maxwell eq.A . G eneration of evanescen t w ave For a p -polarized w ave propagates in the x d irection 2 : d ielectric (z>0) 1 : m etal (z0, 2H =[0 , 2yH , 0 ] )( 22 tzkxki zxe ω−+

2E =[ 2xE , 0 , 2zE ] )( 22 tzkxki zxe ω−+

z

Field intensity within metal

Theory of SPR - Dispersion Relationsubstitute (5) for (8) 1yH - 2yH =0

& (6) 1

1

εzk

1yH +2

2

εzk

2yH =0

∴ 1 -1

1

1

εzk

2

2

εzk = 0

Do the same procedure from (1) & combine the above result, one can get

zik =22)( xi kc

−ωε (9)

xk = cω

21

21

εεεε+

dispersion relation

0 0.002 0.004 0.006 0.008 0.01 0.0120

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

a rbitra ry unit

arbi

trary

uni

t

H2O

H2O -Au

BK7 BK7-Au

1

2

Theory of SPR - Penetration DepthF ro m (9 ) zk i s im a g in a r y ∴ zk = | zk | i

Q E = 0E)( 11 tzkxki zxe ω−−

∴ E = 0E|||| zk ze − , e x p o n e n t ia l d e c a y

p e n e t ra t io n d e p th in m e ta l : ||1

1zk= π

λ2 2

1

21

′+′

ε

εε

p e n e t r a t io n d e p th in d ie le c t r ic : ||1

2zk= π

λ2 22

21

εεε +′

w h e re th e d ie le c t r ic fu n c t io n o f th e m e ta l 1ε =′

1ε + i ″

1ε

-200 0 200 400 600 800 1000 12000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1Penetra tion Depth for Vis ible Light

-30.9 nm 204.3 nm

lamda=539.1 nm

Depth (nm)

Inte

nsity

in Au in die lectric

-200 0 200 400 600 800 1000 12000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1Penetra tion Depth for Near Infrared

-24.0 nm 697.3 nm

lamda=826.6 nm

Depth (nm)

Inte

nsity

in Au in die lectric

SPR spectrum: R vs theta & R vs lamdaB. Excitation of Surface Plasmons by Light ATR Coupler

momentum : cωh

cωh

0ε

xk = 0ε cω sinθ (θ :incident angle)

From Maxwell’s eqs & Boundary Conditions for a p-polarized light: ikr = ( zik / iε - zkk / kε ) / ( zik / iε + zkk / kε ) [ Fresnel’s equations ]

012r =0E

Er =( 01r + 12rαie2 )/(1+ 01r 12r

αie2 )

11 dkz ⋅=α d1 : thickness of the metal film

2012 || rR =

64 66 68 70 72 74 76 78 800

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Incident angle (deg)

lamda=600 nmlamda=750 nm

(BK7 / Au / H2O)

d=48 nm

Ref

lect

ance

550 600 650 700 750 8000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Wavelength (nm)

Ref

lect

ance

deg=72.0 deg=68.5

(BK7 / Au / H2O )d=48 nm

Ei

Z

Er

X

Prism

θ

pε

mε

sε spkSample

KXK

SPR spectrum : different metal

60 62 64 66 68 70 72 74 76 78 800

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

degree

R

lamda=632.8 nm

d=30.0 nm

d=47.5 nm

d=70.0 nm

d=90.0 nm

(BK7/Au/H2O)

40 41 42 43 44 45 46 47 48 490

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

degreeR

lamda=550.0 nm

d=49.0 nm

d=60.0 nm

d=37.0 nm

d=20.0 nmd=89.0 nm

(BK7/Ag/Air)

SPR Spectrum : different n, k, d

65 70 75 80 850

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Incident angle (deg)

Ref

lect

ance

1.41.51.6

d=4 nmk=0.1

n=

lamda=652.6 nm

65 70 75 80 850

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Incident angle (deg)

Ref

lect

ance

lamda=652.6 nm0.10.20.3

k=

n=1.5 d=4 nm

65 70 75 80 850

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Incident angle (deg)

Ref

lect

ance

la mda =652.6 nm4 nm6 nm8 nm

n=1.5 k=0.1

d=

SPR Spectrum : VISIBLE vs NIR

42 43 44 45 46 47 48 49 50 51 520

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

degree

R

lamda=539.1 nm

40 41 42 43 44 45 46 47 48 49 500

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

degreeR

lamda=826.6 nm

4 Layers : Transparent vs Absorbing

37 38 39 40 41 42 43 44 45 46 470

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

degree

R

Quartz / Ag / LiF / Air

0 25 50 100200500

dLiF(A)

37 38 39 40 41 42 43 44 45 46 470

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

degree

R

Quartz / Ag / C / Air

0 23 46 71 97 123149

dC(A)

6 Layers : Biochip configuration

CouplerMetalLinkerLigandAnalyteSolution

60 65 70 75 80 85 900

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

degree

R

BK7 / Au / Linker / Ligand / Ana lyte / Wa te r

Lamda=1152 nm

1. BK7 (n=1.511)2. Au (n=0.330 k=7.930, d=39.2nm)3. Linker (n=1.45, d=50nm)4. Ligand (n=1.42, d=20nm)5. Analyte (n=1.40, d=20nm)6. Water (n=1.322)

60 65 70 75 80 85 900

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

degree

R

BK7 / Au / Linker / Ligand / Ana lyte / Wa te r

Lamda=633 nm

1. BK7 (n=1.515)2. Au (n=0.165 k=3.511, d=48.4nm)3. Linker (n=1.45, d=50nm)4. Ligand (n=1.42, d=20nm)5. Analyte (n=1.40, d=20nm)6. Water (n=1.332)

Surface Plasmon Resonance (SPR)

The n, t, and k values of a dielectrical layer (e.g. proteins) contain information about the amount (mass) of material in the deposited layer. This provides the means for a measurement of the binding parameters of interacting biological molecules, and together with the thickness of the layer, allows an evaluation of the structural arrangement of the molecules which form the film.

(Estimated error ∆n = ± 0.02; ∆t = ± 1; ∆k = ± 0.02)

)]2/()1[(/1.0 22 +−== avav nnAtMdtm

3/)2( 222 spav nnn +=)]2/()1[(/ 22 +−= avav nnAMdVolume Mass Density

Thickness, t (nm) )4/( πλβ ck =

Surface Mass Density

)2)(2/()()(

)]2/()1(//[))((3.022

22

+++=

+−−−=

bb

bbpppbp

nnnnnf

nnVMAnnntfmHeterogeneous mixtures (Lorentz-Lorenz relation)

Spectrum Ambiguity

Not unique (n, d) for Analyte Layer

To Solve the problem:

1. Multi-solvent approach2. Two-color spectroscopy

R

θ

n

d

Near-Infrared Surface Plasmon ResonanceMeasurements of Ultrathin Films

Angle Shift and SPR Imaging Experiments

Analytical Chemistry.1999,71,3928-3934

Bryce P. Nelson, Anthony G. Frutos,Jennifer M. Brockman,and Robert M. CornDepartment of Chemistry,University of Wisconsin-Madison

Application of Surface Plasmon in Microscopic Technique-Surface Plasmon Microscopy(SPM)

Advantages: improve resolution in 1.low-contrast thin-film samples(such as monolayer molecule)2.low index variations

Knoll(1989)

Surface Plasmon Microscopy Image

Monolayer of DMPA(dimyristoylphosphatidic acid)47.20° 47.70°

Fluorescence microscopy

bare silver surface

SiOx coating

Surface Plasmon Resonance Imaging System

Anal. Chem. 2000,72,6003-6009

SPR Imaging Measurements Why NIR ?• Incoherent Light Source avoid

laser fringe• NIR white light/interference

filter increase reflective light intensity SN ratio

• Better Image Contrast due to increase light intensity

• Fixed incident angle and detect reflective intensity by CCD camera

• Differential adsorption measurement on DNA/biopolymer arrays attached on gold surface

filter

SPR Imaging Measurementssingle-stranded DNA binding protein adsorption

Fiber Optic SPR Sensor-A Novel Stride in Sensor Design

From Chip-Based to Optical Fiber-Based

PortabilityFlexibilityRapid AnalysesInterchangeable

Fiber Optic SPR Sensor

• Analogy to Planar Waveguide Device

Fiber Optic SPR Sensor Theoretical Analysis

J. Homola , R. Slavik(1996) • Planar Waveguide Approach & Transfer Matrix Method• Numerical Complex-Zero-Finding Procedure• Calculate Complex Propagation Constant

Simulation Result Experiment Result

Fiber Optic SPR SensorDesign Consideration

• Optic Fiber ConsiderationFiber material-glass fiber , HiBi fiberSingle mode or Multimode-• Sensor Geometry Design

Residual cladding depth(d0)-Polish Side-Tip angle-resonant angle?Metal film thickness

45-75nm