surface-plasmon related ultra sensitive analytical methods and their bio- & nano- applications
DESCRIPTION
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. Outline. Introduction - PowerPoint PPT PresentationTRANSCRIPT
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Surface-plasmon related ultra Surface-plasmon related ultra sensitive analytical methods and sensitive analytical methods and
their bio- & nano- applicationstheir 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.
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Introduction
Surface plasmon fluorescence immunoassays
Bridge SPR technology and nano-world
Development of surface plasmon diffraction sensor
Summary
Outline
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e.g.1. Surface plasmon
resonance (SPR) 2. Quartz crystal
microbalance (QCM)
3. Reflectometry interference spectroscopy (RIfS)
4. Surface plasmon diffraction (SPD)
5. Microcantilever6. ...
e.g. 1. Surface plasmon
fluorescence spectroscopy (SPFS)
2. Total internal reflection fluorescence (TIRF)
3. ...
Labeling Label-free
insufficient sensitivitynon-specific bindingcomplex apparatus
additional time and costs for labelingunnatural bindingnonlinear signal
Biosensors
Mass-labels?Beacon tech?
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Distinctive features of SPR
1. Short range phenomenon
2. Enhanced electromagnetic field
3. Propagating with high attenuation
4. p-polarized
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Principle of Surface Plasmon Fluorescence Spectroscopy (SPFS)
Dipole-to-dipole coupling Surface plasmonBack-coupling
Free emission
prism
metal
dye
dielectric
Prism Au water
0 100
0
5
10
15
20
Flu
ore
sce
nce
Yie
ld %
Fie
ld-e
nh
an
cem
en
t F
act
or
z /nm
0
100
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laser 632.8 nm
polarizers
photodiode
flow cell filter
2
goniometer
lock-in amplifier
photon-counter
motor-steering
PC
chopper
Laser-shutter
attenuatorlens
shutter controller
prism
The set-up
a) PMT b) FOSc) CCD camera
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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…
Good chemistry (for NSB, activation, regeneration…)
Lateral control of ligand density
Compatible with the physics of the biosensor
Surface matrix for biosensing
Ideally…
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: antibody
: fluorophore
: dextran
: SAM
Au water
0 50 100
0
5
10
15
20
Fluo
resc
ence
Yie
ld %
Fiel
d-en
hanc
emen
t Fac
tor
z /nm 0
100
: streptavidin
2D (e.g. layer-by-layer assembly)
3D (e.g. dextran matrix)
Interfacial design of sensing matrix
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45 50 55 60
0.0
0.3
0.6
0.9
a
ib*
J*
Angle /degrees
Re
fleci
tivity
R
0.0
0.5
1.0
i->j*a->b*
Flu
ore
sce
nce
/ 1
0 4 c
ps
40 50 600.0
0.4
0.8
abcdefghijk
Re
flect
ivity
R
Angle /degrees
0 5 10
57
58
59
60
kj*
ih
gf
ed
cb*
a
SP
R a
ngle
/deg
rees
Layer
LbL to clarify metal-induced quenching
IJ* : Ib* = 34Alternating biotin-IgG and SA, decorate certain layer by Alexa fluor labeled SA
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Surface preparation for dextran matrix
0 10 20 30 400.0
0.1
0.2
0.3
0.4
0.5
0.6
Re
flect
ivity
R
Time /minutes
(1) (2) (3)
(1)
(2)
(3)
~8 ng mm-2
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0 20 40 600.6
0.9
1.2a
b
c
d
e
Flu
ores
cenc
e /1
0 4 c
ps
Time /minutes0 200 400 600
1
2
3
333 fM
buffer
3.3 pM
33 pM
67 fM
333 fM
Flu
ores
cenc
e /1
0 4 c
ps
Time /minutes
Limit of detection (LOD) evaluation under mass-transport limited binding
condition
Baseline deviation bulkm Akdt
ABd
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45 50 55 600
10
20
30
40
50
60
70
80
90
Angle /degrees
SP
R R
%0
1
2
3
4
5
6
7
8
0.00 0.08 0.16
0
3
6
Fluo
resc
ence
/10
9 cps
SPR angle shift /degree
ed
c
ba
abcde
e
d
c
a
b
Flu
ores
cenc
e / r
1*r 2*
105 c
ps
LOD at atto-molar level
Correlation between SPR and
fluorescence
Translate the LOD level to molecular surface concentration~10 molecules/(mm2*min)
10-2 100 102 104 106100
103
106
Baseline deviation level
Bin
din
g s
ign
al /
cps
min-1
Concentration /fM
Yu, F., Persson, B., Loefas, S., Knoll, W. JACS, 126, 8902 -8903, 2004.
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-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2-1000
0
1000
2000
3000
4000
5000
6000
7000
NS
B o
f pl
asm
a
PSA sample in buffer PSA sample in plasma
Flu
ores
cenc
e in
crem
ent/c
ps
Concentration /pM
slope km= 0.98(D/h)2/3(v/bx)1/3 -2/3
Prostate-specific antigen (PSA) sandwich assay
D=kT/6 a
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0 20 40 60 80
0.1
0.2
0.3
0.4
0.5
Time /minutes
SP
R R
efle
ctiv
ity
5
10
15
20
bufferrinse
plasma with 0.5 mg/mL free dextran
detectionAb in buffer
Flu
ores
cenc
e /1
0 3 c
ps
0.1 1 10 100 10000.1
1
10
100
1000
LOD=80 fM
baseline level
Concentration of PSA /pM
LOD of PSA assay without plasma NSB
Yu, F., Persson, B., Loefas, S., Knoll, W. ANALYTICAL CHEMISTRY, in press.
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125 nm
Streptavidin-latex bead in SPR sensing
SA doping ratio:
~300 SA per bead
Utilities:
1, Signal amplification
2, Introduce surface scattering
3, Being functional matrix itself (2.5
D)
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300 400 500 600 700 800 900 1000 1100
0
200
400
600
633 nm
Inte
nsity
/A.U
.
Wavelength /nm
Au
Biotin SAM (1:9)
SA-Lx
FOS (Fiber optic spectrometer)
Surface plasmon enhanced light-
scattering633 nm laser
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45 50 55 60 650
20
40
60
80
100 (1) (2) (3) (4) (5)
Ligh
t sca
tterin
g /A
.U.
SP
R
Angle /degrees
0
200
400
600
800
1000
(1) (2) (3) (4) (5)
Coverage dependent scattering
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One step SPR detection of 15mer oligonucleotide by latex-amplification
-5 0 5 10 15 20 250.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
LOD = mean + 3 sd (N.C.)
LOD = ~2 pM
Bin
din
g S
lop
e /
A.U
.
Concentration /pM
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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)
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
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Color Multiplexed hybridization detection test
+QD655-T1
(MM0 for P1)
+QD565-T2
(MM0 for P2)
Microarray image from SPM
P1 P2
P3P1+P2
P1 P2
P3P1+P2
P1 P2 P1 P2
Excitation-Filter (543nm)
Robelek, R., Niu, L., Schmid, E. L., Knoll, W. ANALYTICAL CHEMISTRY, in press
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Dielectric grating
01
2
-2-1 Diffraction
orders (m)
gmkk PSPmdiff
Au
/2g
Principle of SPDS
nd, the grating amplitude
2
0
nd
II d
Functional areaNonfunctional area
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TIR diffraction vs. SPR diffraction
01
2
-2-1
01
2
-2-1
TIR mode ATR/SPR mode
Polystyrene pattern 92 94 96 98
0
5
10
15
2-2
-1
1
0 Au (ATR) glass (TIR)
Diff
ract
ion
Inte
nsity
A.U
.
Angle [o]
Au
glass
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Diffraction patterns
Surface plasmon microscopy images
Diffraction photographs
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PDMS
Si
Si
Si
PDMS
PDMS
Au
Au
AuPDMS
Photoresist
Photoresistpattern
1
3
2
5
7
6
4
Micro-contact printing for SAM patterning
Functional SAM
Nonfunctional SAM
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0 50 100 150
0.00
0.50
1.00
Diff
ract
ion
Inte
nsity
/mV
Time /minutes
57.2 57.4 57.6 57.8-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
quadratic fit
Diff
ract
ion
inte
nsity
/mV
SPR minimum angle /degrees
Quadratic property of the diffraction signal
Bio
tin S
AM
Anti-biotin antibody
2
0
nd
II d
Yu, F., Tian, S., Yao, D., Knoll, W. ANALYTICAL CHEMISTRY, 76, 3530 -3535, 2004.
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0 5 10 15 20 25 301.04
1.06
1.08
43 oC32 oC 22 oC22 oC22 oC
Diff
ract
ion
/mV
Time /minutes
0.22
0.24
SP
R R
Self-referencing property of the diffraction sensor- a temperature variation test
Yu, F., Knoll, W. ANALYTICAL CHEMISTRY, 76, 1971-1975, 2004.
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0.1 1 10
1E-4
1E-3
0.01
baseline deviation level
Bin
ding
rat
e /m
V1
/2m
in-1
Concentration of hCG /nM
SPDS for label-free detection of human
chorionic gonadotropin (hCG)
0 50 100 150
0.00
0.05
0.10
0.4buffer rinse
(d)
(c)(b)
(a)
Cor
rect
ed d
iffra
ctio
n in
tens
ity
chan
ge/m
V1/
2
Time /minutes
(a) 500 nM Fab-biotin(b) 50 nM hCG(c) 50 nM hCG in 1 mg/mL BSA (d) 1 mg/mL BSA
SA
MS
AF
ab
hC
G
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-10 0 10 20 30 40 50 60 70 80
0.0
0.2
0.4
0.6
0.8
rinse
DNA probe
rinse
SA
Cor
rect
ed D
I /(m
V)1/
2
Diff
ract
ion
inte
nsity
/mV
Time /minutes
0.0
0.3
0.6
0.9
functional nonfunctional
bio
tin S
AM
probe DNA
SA
target DNA
SPDS for oligonucleotide detection1, surface preparation
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SPDS for oligonucleotide detection2, kinetic analysis
-10 0 10 20 30 40 50 60 70-0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
T15-MM2
T15-MM1
T15-MM0
regenerationby NaOH
Co
rre
cte
d
DI /
(mV
)1/2
Time /minutes
Name HE* koff (s-1) kon (M-1s-1) KD (M)
T15-MM0 84% 1.310-4 6.6104 210-9
T15-MM1 62% 1.110-3 2.4104 4.610-8
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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10-3 10-2 10-1 100 101 102 103 104 105-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
T15 MM0
T15 MM1
R
eq /A
.U.
Concentration c0 /nM
SPDS for oligonucleotide detection3, adsorption isotherm analysis
Deq Kc
RcR
0
max0
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
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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)