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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
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 (z<0)
z>0, 2H =[0 , 2yH , 0 ] )( 22 tzkxki zxe ω−+
2E =[ 2xE , 0 , 2zE ] )( 22 tzkxki zxe ω−+
z<0, 1H =[0 , 1yH , 0 ] )( 11 tzkxki zxe ω−−
1E =[ 1xE , 0 , 1zE ] )( 11 tzkxki zxe ω−−
M a x w e ll’s e q s .
×∇ iE =t
Hc
i
∂∂− 1 (1 )
×∇ iH =t
Ec
ii ∂
∂− 1ε (2 )
•∇ iε iE = 0 (3 )
•∇ iH = 0 (4 )
C o n tin u i ty r e la t io n s (b o u n d a r y c o n d it io n s ) 1xE = 2xE (5 )
1yH = 2yH (6 )
1ε 1zE = 2ε 2zE (7 )
(2 ) z
H yi
∂∂
= - i iε cω
xiE
1zk 1yH =cω
1ε 1xE
2zk 2yH = -cω
2ε 2xE (8 )
Incident light Totally internallyreflected light
Evanscent fieldregion d
pMedium n2
Medium n1
exponentially
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 = πλ
2 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 = πλ
2 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 + 12r αie2 )/(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( 222spav 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
