Surface-plasmon related ultra sensitive analytical methods and their bio-

1
Surface-plasmon related ultra sensitive
analytical methods and their bio- nano-
applications

• Fang Yu, Wolfgang Knoll
• Max-Planck Institute for Polymer Research, Mainz,
  Germany

International Congress of Nanotechnology, Nov.
7-10, 2004, San Francisco, USA.
2
Outline

• Introduction
• Surface plasmon fluorescence immunoassays
• Bridge SPR technology and nano-world
• Development of surface plasmon diffraction
  sensor
• Summary

3
Biosensors
additional time and costs for labeling unnatural
binding nonlinear signal

• e.g.
• Surface plasmon resonance (SPR)
• Quartz crystal microbalance (QCM)
• Reflectometry interference spectroscopy (RIfS)
• Surface plasmon diffraction (SPD)
• Microcantilever
• ...

• e.g.
• Surface plasmon fluorescence spectroscopy (SPFS)
• Total internal reflection fluorescence (TIRF)
• ...

Labeling
Label-free
Mass-labels? Beacon tech?
insufficient sensitivity non-specific
binding complex apparatus
4
Distinctive features of SPR

1. Short range phenomenon
2. Enhanced electromagnetic field
3. Propagating with high attenuation
4. p-polarized

5
Principle of Surface Plasmon Fluorescence
Spectroscopy (SPFS)
Prism Au
water
Dipole-to-dipole coupling
Surface plasmon Back-coupling
Free emission

20
prism
15
100
metal
10
Field-enhancement Factor
Fluorescence Yield
dye
5
dielectric
0
0
0
100
z /nm
6
The set-up
photodiode
2?
Laser-shutter
laser 632.8 nm
goniometer
prism
?
lens
chopper
polarizers
attenuator
flow cell
filter
a) PMT b) FOS c) CCD camera
PC
lock-in amplifier
photon- counter
motor- steering
shutter controller
7
Surface matrix for biosensing

• Good chemistry (for NSB, activation,
  regeneration)
• Lateral control of ligand density
• Compatible with the physics of the biosensor

Ideally

• 2D (SAM, lipid bilayer)
• 2.5D (Layer-by-layer, nano-particle,
  nano-capsule, nano-wire modified surface, porous
  or roughened surface)
• 3D (brush type polymer, hydrogel network, plasma-
  or electro-polymerized matrix)

Dimension
8
Interfacial design of sensing matrix
Au
water
antibody
2D (e.g. layer-by-layer assembly)
fluorophore
streptavidin
3D (e.g. dextran matrix)
SAM
dextran
9
LbL to clarify metal-induced quenching
Alternating biotin-IgG and SA, decorate certain
layer by Alexa fluor labeled SA
IJ Ib 34
10
Surface preparation for dextran matrix
(2)
(3)
(1)
(3)
(2)
8 ng mm-2
(1)
11
Limit of detection (LOD) evaluation under
mass-transport limited binding condition
Baseline deviation
12
Correlation between SPR and fluorescence
LOD at atto-molar level
Translate the LOD level to molecular surface
concentration 10 molecules/(mm2min)
Yu, F., Persson, B., Loefas, S., Knoll, W. JACS,
126, 8902 -8903, 2004.
13
Prostate-specific antigen (PSA) sandwich assay
slope? km 0.98(D/h)2/3(v/bx)1/3 ? ? -2/3
14
LOD of PSA assay without plasma NSB
Yu, F., Persson, B., Loefas, S., Knoll, W.
ANALYTICAL CHEMISTRY, in press.
15
Streptavidin-latex bead in SPR sensing
SA doping ratio 300 SA per bead
125 nm
Utilities 1, Signal amplification 2, Introduce
surface scattering 3, Being functional matrix
itself (2.5 D)
16
FOS (Fiber optic spectrometer)
SA-Lx
Biotin SAM (19)
Au
Surface plasmon enhanced light-scattering
633 nm laser
17
Coverage dependent scattering
18
One step SPR detection of 15mer oligonucleotide
by latex-amplification
19
DNA conjugated core/shell QDs
Core/shell QDs supplied by Q-Dot Corp. high
stability in PBS before and after
conjugation wavelength 565nm (green),
585nm (yellow), 605nm (orange) and 655nm
(red) are all excitable with 543nm (green
laser)
5-biotinylated target DNA
20
Color Multiplexed hybridization detection test
Excitation-Filter (543nm)
QD565-T2 (MM0 for P2)
QD655-T1 (MM0 for P1)
Microarray image from SPM
P2
P1
Robelek, R., Niu, L., Schmid, E. L., Knoll, W.
ANALYTICAL CHEMISTRY, in press
21
Principle of SPDS
-2
-1
Diffraction orders (m)
0
1
2
Functional area
Nonfunctional area
Au
Dielectric grating
?nd, the grating amplitude
?
22
TIR diffraction vs. SPR diffraction
-2
-1
0
1
2
-2
-1
0
?
1
?
2
glass
Au
?
Polystyrene pattern
TIR mode
ATR/SPR mode
23
Diffraction patterns
Surface plasmon microscopy images
Diffraction photographs
24
Micro-contact printing for SAM patterning
4
Photoresist
PDMS
Si
1
5
Photoresist pattern
PDMS
Si
Au
2
6
PDMS
Si
Au
3
Functional SAM
7
Nonfunctional SAM
PDMS
Au
25
Quadratic property of the diffraction signal
Anti-biotin antibody
Biotin SAM
Yu, F., Tian, S., Yao, D., Knoll, W. ANALYTICAL
CHEMISTRY, 76, 3530 -3535, 2004.
26
Self-referencing property of the diffraction
sensor - a temperature variation test
Yu, F., Knoll, W. ANALYTICAL CHEMISTRY, 76,
1971-1975, 2004.
27
hCG
SPDS for label-free detection of human
chorionic gonadotropin (hCG)
Fab
SA
SAM
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SPDS for oligonucleotide detection 1, surface
preparation
target DNA
probe DNA
SA
biotin SAM
nonfunctional
functional
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SPDS for oligonucleotide detection 2, kinetic
analysis
Name HE koff (s-1) kon (M-1s-1) KD (M)
T15-MM0 84 1.3?10-4 6.6?104 2?10-9
T15-MM1 62 1.1?10-3 2.4?104 4.6?10-8
T15-MM2 0 N/A N/A N/A
HE Hybridization efficiency
Yu, F., Yao, D., Knoll, W. NUCLEIC ACIDS
RESEARCH, 32, e75, 2004.
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SPDS for oligonucleotide detection 3, adsorption
isotherm analysis
KD0
KD1
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Summary

• Ultra-sensitive SPFS immunoassay is established
  with the aid of three-dimensionally extended
  matrix
• Initial attempts of SPR based nano-sensing
• SPDS is developed for label-free analysis of
  protein interactions and oligonucleotide
  hybridizations

32
Acknowledgements
Neal Armstrong (University of Arizona) Akira Baba
(University of Texas at Houston ) Shengjun Tian
(MPIP) Lau King Hang Aaron (IMRE, Singapore)
(for helps in the diffraction work) Björn
Persson (Biacore) Stefan Löfås (Biacore) Renate
Sekul (Graffinity) Holger Ottleben (Graffinity)
(for collaborations) Danica Christensen
(MPIP) (for the LBL work) Pierre Thiébaud
(MPIP) Darick Ding (MPIP) (for the set-up
engineering ) Danfeng Yao (MPIP) Thomas Neumann
(Graffinity) Eva - Kathrin Sinner (MPI
biochemistry) Peter E. Nielsen (Panum Institute,
Denmark) Keiko Tawa (AIST Osaka) Rudi Robelek
(IMRE, Singapore) Lifang Niu (IMRE,
Singapore) (for the DNA/QDs part)