Transcript Supramolecular interfacial architectures for biosensing

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
 Surface plasmon fluorescence immunoassays
 Bridge SPR technology and nano-world
 Development of surface plasmon diffraction sensor
 Summary
Biosensors
additional time and costs for labeling
unnatural binding
nonlinear signal
e.g.
1. Surface plasmon
e.g.
resonance (SPR)
1. Surface plasmon
2. Quartz crystal
fluorescence
microbalance
spectroscopy
Mass-labels?
(QCM)
Labeling
Label-free
(SPFS)
Beacon tech?
3. Reflectometry
2. Total internal
interference
reflection
spectroscopy
fluorescence
insufficient sensitivity
(RIfS)
(TIRF)
non-specific binding
4. Surface plasmon
3. ...
complex apparatus
diffraction (SPD)
5. Microcantilever
6. ...
Distinctive features of SPR
1. Short range phenomenon
2. Enhanced electromagnetic field
3. Propagating with high attenuation
4. p-polarized
Principle of Surface Plasmon Fluorescence
Spectroscopy (SPFS)
Au
Dipole-to-dipole coupling
water
20
prism
15
100
10
5
0
0
0
100
z /nm
Fluorescence Yield %
Field-enhancement Factor
Prism
metal
dye
dielectric
Surface plasmon
Back-coupling
Free emission
The set-up
photodiode
Laser-shutter
laser 632.8 nm
prism
2
goniometer

chopper
polarizers
flow cell
lens
attenuator
filter
a) PMT
b) FOS
c) CCD camera
PC
shutter
controller
motorsteering
photoncounter
lock-in
amplifier
Surface matrix for biosensing
Ideally…

Good chemistry (for NSB, activation,
regeneration…)

Lateral control of ligand density

Compatible with the physics of the
biosensor

2D (SAM, lipid bilayer…)

2.5D (Layer-by-layer, nano-particle, nanocapsule, nano-wire modified surface,
porous or roughened surface…)

3D (brush type polymer, hydrogel
network, plasma- or electro-polymerized
matrix…)
Dimension…
Interfacial design of sensing matrix
Au
water
15
100
10
5
z /nm
0
0
: antibody
: fluorophore
50
Fluorescence Yield %
Field-enhancement Factor
20
0
100
2D (e.g. layer-by-layer
assembly)
: streptavidin
: SAM
: dextran
3D (e.g. dextran matrix)
LbL to clarify metal-induced quenching
0.9
0.8
0.4
59
58
c
b*
57
a
0
d
e
f
g
h
i
j*
a
b
c
d
e
f
g
h
i
j
k
k
Layer
5
0.6
4
Reflecitivity R
60
SPR angle /degrees
Reflectivity R
1.0
0.3
0.5
a->b* i->j* i
0.0
b* 0.0
a
10
0.0
40
50
Angle /degrees
60
Alternating biotin-IgG and SA, decorate
certain layer by Alexa fluor labeled SA
Fluorescence / 10 cps
J*
45
50
55
Angle /degrees
IJ* : Ib* = 34
60
Surface preparation for dextran matrix
0.6
(3)
0.5
Reflectivity R
(1)
(2)
0.4
0.3
~8 ng mm-2
(1)
0.2
0.1
0.0
0
10
20
Time /minutes
30
40
(2)
(3)
Limit of detection (LOD) evaluation under
mass-transport limited binding condition
33 pM
4
a
Fluorescence /10 cps
1.2
4
Fluorescence /10 cps
3
b
c
0.9
d
e
buffer
2
3.3 pM
333 fM
333 fM
1
67 fM
0.6
0
20
40
Time /minutes
Baseline deviation
60
0
200
400
Time /minutes
d AB 
 k m Abulk 
dt
600
Correlation between
SPR and fluorescence
LOD at atto-molar level
8
7
-1
3
10
50
40
30
20
Baseline deviation level
0
10
-2
10
0
2
4
10
10
10
Concentration /fM
6
10
cps
9
60
d
Fluorescence /10
10
SPR R %
Binding signal /cps min
70
6
d
5
c
3
0
6
e
6
a
c
3
0.00 0.08 0.16
SPR angle shift /degree
b
0
a
50
2
abcde
10
45
4
b
55
1
0
5
e
80
Fluorescence / r 1*r2*10 cps
90
60
Angle /degrees
Translate the LOD level to molecular surface concentration
~10 molecules/(mm2*min)
Yu, F., Persson, B., Loefas, S., Knoll, W. JACS, 126, 8902 -8903, 2004.
Prostate-specific antigen (PSA) sandwich assay
7000
5000
4000
3000
2000
1000
0
-1000
NSB of plasma
Fluorescence increment/cps
6000
-0.2
PSA sample in buffer
PSA sample in plasma
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Concentration /pM
slope km= 0.98(D/h)2/3(v/bx)1/3   -2/3
D=kT/6 a
LOD of PSA assay without plasma NSB
detection
Ab in buffer
plasma with
0.5 mg/mL
free dextran
buffer
rinse
15
0.3
10
0.2
5
0.1
1000
3
0.4
20
Fluorescence /10 cps
SPR Reflectivity
0.5
100
10
LOD=80 fM
1
baseline level
0.1
0
20
40
60
Time /minutes
80
0.1
1
10
100
1000
Concentration of PSA /pM
Yu, F., Persson, B., Loefas, S., Knoll, W. ANALYTICAL CHEMISTRY, in press.
Streptavidin-latex bead in SPR sensing
SA doping ratio:
125 nm
~300 SA per bead
Utilities:
1, Signal amplification
2, Introduce surface scattering
3, Being functional matrix itself (2.5 D)
Intensity /A.U.
600
633 nm
400
200
0
300
400
500
600
700
800
900
1000
1100
Wavelength /nm
FOS (Fiber optic spectrometer)
SA-Lx
Biotin SAM (1:9)
Au
Surface plasmon
enhanced light-scattering
633 nm laser
Coverage dependent scattering
100
SPR
80
1000
800
60
600
40
400
200
20
0
0
45
50
55
60
65
Angle /degrees
(1)
(2)
(3)
(4)
(5)
Light scattering /A.U.
(1)
(2)
(3)
(4)
(5)
One step SPR detection of 15mer
oligonucleotide by latex-amplification
1.8
1.6
Binding Slope /A.U.
1.4
1.2
1.0
LOD = ~2 pM
0.8
0.6
LOD = mean + 3 sd (N.C.)
0.4
0.2
-5
0
5
10
15
Concentration /pM
20
25
DNA conjugated core/shell QDs
Core/shell QDs supplied by QDot Corp. :
high stability in PBS before
and after conjugation
5’-biotinylated
target DNA
wavelength 565nm (green),
585nm (yellow), 605nm
(orange) and 655nm (red)
are all excitable with 543nm
(green laser)
Color Multiplexed hybridization detection test
Excitation-Filter
(543nm)
+QD565-T2
(MM0 for P2)
Microarray image
from SPM
P1
P2
P1
P2
P1+
P2
P3
P1+
P2
P3
P1
P2
P1
P2
+QD655-T1
(MM0 for P1)
Robelek, R., Niu, L., Schmid, E. L., Knoll, W. ANALYTICAL CHEMISTRY, in press
Principle of SPDS
-2
Diffraction
orders (m)
-1
0
1
2
Functional area
Nonfunctional area
Au
Dielectric grating

m


kdiff  kPSP  mg

g  2 / 
nd, the grating amplitude
  nd 

I d  I 0 




2
TIR diffraction vs. SPR diffraction
-2
-1
1
2
-2

glass
-1

0
1
2
Au

Polystyrene pattern
Diffraction Intensity A.U.
0
0
15
Au (ATR)
glass (TIR)
-1
10
1
5
2
-2
0
92
94
96
o
Angle [ ]
TIR mode
ATR/SPR mode
98
Diffraction patterns
Surface plasmon
microscopy images
Diffraction photographs
Micro-contact printing for SAM patterning
4
Si
Photoresist
1
PDMS
5
Si
Photoresist
pattern
PDMS
Au
2
6
PDMS
Si
Au
3
Functional SAM
Nonfunctional SAM
7
PDMS
Au
Quadratic property of the diffraction signal
Anti-biotin antibody
Biotin SAM
  nd 

I d  I 0 
  
1.4
Diffraction intensity /mV
1.00
Diffraction Intensity /mV
2
0.50
0.00
0
50
100
Time /minutes
150
1.2
quadratic fit
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
57.2
57.4
57.6
57.8
SPR minimum angle  /degrees
Yu, F., Tian, S., Yao, D., Knoll, W. ANALYTICAL CHEMISTRY, 76, 3530 -3535, 2004.
Self-referencing property of the diffraction sensor
- a temperature variation test
SPR R
0.24
0.22
1.08
o
o
Diffraction /mV
22 C
32 C
o
o
o
22 C
43 C
22 C
1.06
1.04
0
5
10
15
20
25
30
Time /minutes
Yu, F., Knoll, W. ANALYTICAL CHEMISTRY, 76, 1971-1975, 2004.
hCG
buffer rinse
0.01
1/2
(a)
-1
0.4
Binding rate /mV min
Corrected diffraction intensity
1/2
change/mV
SAM
SA
Fab
SPDS for label-free
detection of human
chorionic gonadotropin (hCG)
0.10
(b)
(c)
0.05
(a)
(b)
(c)
(d)
(d)
0.00
0
50
100
Time /minutes
500 nM Fab-biotin
50 nM hCG
50 nM hCG in 1 mg/mL BSA
1 mg/mL BSA
150
1E-3
1E-4
baseline deviation level
0.1
1
10
Concentration of hCG /nM
SPDS for oligonucleotide detection
1, surface preparation
biotin SAM
functional nonfunctional
SA
rinse
0.4
0.6
DNA probe
0.2
0.3
0.0
0.0
-10
0
10
20
30
40
50
Time /minutes
60
70
80
1/2
0.9
rinse
0.6
Corrected DI /(mV)
target DNA
SA
probe DNA
Diffraction intensity /mV
0.8
SPDS for oligonucleotide detection
2, kinetic analysis
0.06
0.05
T15-MM0
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
Corrected DI /(mV)
1/2
0.04
T15-MM1
0.03
regeneration
by NaOH
0.02
0.01
T15-MM2
0.00
-0.01
-10
0
10
20
30
40
50
60
70
HE: Hybridization efficiency
Time /minutes
Yu, F., Yao, D., Knoll, W. NUCLEIC ACIDS RESEARCH, 32, e75, 2004.
SPDS for oligonucleotide detection
3, adsorption isotherm analysis
1.2
1.0
Req /A.U.
0.8
T15 MM0
0.6
0.4
c0 Rmax
Req 
c0  K D
T15 MM1
0.2
0.0
KD0
-0.2
-3
10
-2
10
-1
10
KD1
0
10
1
10
2
10
3
10
Concentration c0 /nM
4
10
5
10
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
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)