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Development of plasmonics-based methods for biosensing
Andrei V. KabashinLP3 UMR 6182 CNRS – Université de la Méditerranée,
Marseille, France
NGC, Hamilton, Canada, 13 August 2009
Peter I. NikitinGeneral Physics Institute of Russian Academy of
Sciences, Moscow, Russia
Alexander GrigorenkoUniversity of Manchester, Manchester, UK
People participated in the project
Sergiy Patskovsky, Michel Meunier, Mathieu Maisonneuve, In-Hyouk Song, M. Skorobogatiy, Galina Nemova, Raman Kashyap, Gregory De Crescenzo
École
Polytechnique
de Montréal, Montreal, Canada
Paras N. Prasad, Przemyslaw Markowicz, Alexander Baev, Wing-Cheung LawInstitute of Lasers, Photonics and Biophotonics, State University of New York at
Buffalo, Buffalo, USA
Biosensing
deals with selectivebinding (recognition) events onthe surface:Antigen –
Antibody,DNA –
DNA
capture,Ligand
–
protein,Protein -protein etc.
Conventional transduction: fluorescent labeling
Biosensing
Disadvantages- The procedure is slow and takes several steps-
Only the result of a reaction can be determined (yes/no)
The index of refraction of most biomaterials is around 1.4-1.44, while the refractive index of water is 1.33
Optical transduction biosensingBinding or recognition events on the surface must be accompanied by a change of refractive index of thin layer (from several nm to hundreds of microns) near the surface
How to detect a small change of refractive index in a thin film???
Thin film interferometry(G. Gauglitz
et. al.)
Wavelength λ Time
λminR
White light
Dielectric waveguides (Lukosz
et. al. 1990)Surface Plasmon Resonanceemploying metal structures(Lindberg et.al., 1983)
Surface plasmon polaritons (SPP) are electromagnetic waves, which propagate over a metal/dielectric interface
SPR angle is extremely sensitive to Δ
n3
Dip is due to SPR
In the resonance ( correct angle of incidence at a given wavelength ), the photon energy is absorbed in a plasmon excitation, resulting in a sharp minimum in the reflected light as a function of angle of incidence
SPR Phenomenon
Surface Plasmon Resonance (SPR): sensing effect
Conditions of the plasmon excitation are extremely (resonantly) sensitive to the dielectric constant of the adjacent medium within a thin layer (200-300 nm).
z
EZ
200 nm
Surface Plasmon Resonance (SPR): sensing effect
Immobilization of a reactant on the gold surface
Conditions of the plasmon excitation are extremely (resonantly) sensitive to the dielectric constant of the adjacent medium within a thin layer (200-300 nm).
Surface Plasmon Resonance (SPR): sensing effectConditions of the plasmon excitation are extremely (resonantly) sensitive to the dielectric constant of the adjacent medium within a thin layer (200-300 nm).
Reaction between reactant and its selective partner Δ
n3Angle (frequency)
shift
SPR technology
SPR biosensing SPR imaging
in vitro (SPR microscopy)
Converging beam Parallel beam
1. Label-free detection (no chromophoric
group or labeling is required)2. Real-time measurements
1. Record resolution of the thickness of organic films on gold (lateral resolution of the order of micron)
2. Multi-channel sensing (gene chips, high throughput screening)Fast Analyses:
Only few minutes are required toobtain reaction kinetics constants
PLASMONICS BIOSENSING PROJECT
Improvement of sensor sensitivity
Miniaturization and cost reduction
Development of novel
nanoplasmonics architectures
Sub-project I: Improvement of sensor
sensitivity
In terms of the refractive index change:Δnmin = (3-10)*10-6
Detection limit of conventional SPR biosensor units
1 pg⋅mm-2 of biomaterial accumulating at the biosensor surface
This sensitivity is satisfactory to study many interactions such as: antibody-antigen, protein-DNA, DNA-DNA, receptor-ligand, etc.
However, this sensitivity needs to be improved for: - Detection of low molecular weight analytes (drugs, some proteins) with mass less than 1,000 Daltons - Detection of extremely small concentrations of larger analytes (antigens etc.), pathogenic at ultra-low levels (detection of deadly virus and bacteria, dangerous at ultra-low concentrations)
How can we improve the sensitivity of SPR biosensing???
Phase properties of light
Amplitude and phase characteristics
I (θ, λ)
φ
(θ, λ)
Amplitude (Intensity) characteristics are related to the length of electric field vector, while phase characteristics are related to rotation of this vertor
50.0 52.5 55.0 57.5 60.0-270
-180
-90
0
90
180
20nm30nm40nm
48.6nm
50nm60nm70nm
Phas
e[de
g.]
Angle[deg.]
45 50 55 60 65-0.10.00.10.20.30.40.50.60.70.80.91.0
70nm50nm30nm
Inte
nsity
Angle[deg.]
Intensity and phase of light reflected under SPRReflected intensity
Phase
Phase of light can experience a sharp jump
under SPR (!!!) A.V. Kabashin, P.I. Nikitin Quant. Electron., 27, 653 (1997); Opt. Commun, 150, 5 (1998)
1.330 1.332 1.334 1.336 1.338 1.340
-100
-50
0
50
100
Phas
e [d
eg.]
Refractive index
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Reflectivity
Phase can be more sensitive to refractive
index change
Methods for phase measurements are extremely
sensitive
+
A.V. Kabashin and P.I. Nikitin, Quant. Electr. 27, 653 (1997); Optics Commun. 150, 5 (1998).PCT Patent WO9857149 (1998), US6628376 (2003)
Concept of Surface Plasmon Resonance Interferometry1. Zernike contrast
mode2. Fringe Mode
0
π
Mach-Zehnder geometry
The concept can equally work with other interferometry geometries under SPR (in partucular, using s-polarized light component as a reference one)
A.N. Grigorenko, P.I. Nikitin and A.V. Kabashin, Appl. Phys. Lett., 75, 3917 (1999)
SPR Interferometry: estimation of sensitivity
Ar
ou N2
: Difference
between
indices of
refraction: Δn
≅1.5⋅10-5
Detection
limit
of
the
SPR interferometry: Δnmin
= 4⋅10-8
(absolute
record for thin
films)
Almost
100-fold more sensitive than
conventional
SPR sensors!!!
A.V. Kabashin and P.I. Nikitin, Optics Commun. 150, 5 (1998).
0 5 0 0 0 10000 15000 20000
0
Δϕ
t(sec)
N2
A r
N2
Ar
N2
ArAr Ar
N 2
Interferometric SPR imaging
Interferometric
SPR imaging
:The
droplet
is
clearly
visible
Zernike contrast mode Fringe mode Conventional
SPR microscopy: no
signal
Images of
ultra-thin
film of
fatty
acide on the
gold surface
Due to higher phase sensitivity, interferometric
SPR imaging enables to detect objects, which are not visible by conventional SPR
A.N. Grigorenko, P.I. Nikitin and A.V. Kabashin, Appl. Phys. Lett., 75, 3917 (1999)
edge
Image of a thin Si film (5 A) on gold
Conventional
SPR: No signal
Interferometric
SPR imagingThe edge of the film is visible by bending of interference fringes
A.N. Grigorenko, P.I. Nikitin and A.V. Kabashin, Appl. Phys. Lett., 75, 3917 (1999)
Example of higher sensitivity of SPR Interferometry
interferometric
SPR image of a Si test–structure with the cell dimensions 100x100 μm2
and thickness of 2 nm
Interferometric SPR imaging: potential for the development of multi-channel arrays (e.g., gene chips)
A.N. Grigorenko, P.I. Nikitin and A.V. Kabashin, Appl. Phys. Lett., 75, 3917 (1999)
Tasks and challenges in applying phase-sensitive methods in SPR
1. Maximization of physical sensitivity (quality of gold film etc.)
2. Maximization of instrumental sensitivity
3. Maximization of dynamic range of phase-sensitive measurements
4. Minimization or elimination of external noises (inert and temperature drifts)
6. Miniaturization and cost reduction
Maximization of physical sensitivity
1.30 1.32 1.34 1.36 1.38 1.40-270
-180
-90
0
90
180
48nm40nm
50nm
Phas
e[de
g.]
Refractive index
In theory, the sensitivity can be infinitively high if the gold layer thickness is well optimized
In practice, the sensitivity is limited by the quality of gold film (thickness, roughness, uniformity) and properties of layers
Lower is the intensity in the SPR, higher is the phase sensitivity
If the deposition conditions are well optimized, you can have the detection limit of Δnmin
= 10-8
and
even
lower
68 69 70 71 72 73 74 7540
50
60
70
80
90
100
110
120
130
140
Ag+Au
Au
Ag
Angle=69.044S = 1.455*104
Angle=72.59S = 7.385*103
Phas
e di
ffere
nce
(p-s
)
Angle [deg]
One of ways to improve the sensitivity is the use of Ag instead of gold
Maximization of instrumental sensitivity
Minimization of instrumental noises, improvement of signal/noise ration in phase-sensitive measurements
SPR interferometry
Main approaches for phase- sensitive measurements
SPR polarimetry
SPR Interferometry
Mach-Zehnder
geometry
-
Information on phase is extracted from spatial interferometry
pattern
-
optical treatment of the resulting signal
Advantages:
-
enables easy lateral resolution (important for gene chips…)
-
gives pure phase-related signal
Common
path
interferometry
SPR Polarimetry
• Information on s and
p polarization• S-polarization
as reference
for measurement• Electronical
signal processing
Advantages:
-
Easy removal of noises and filtering by processing electronics
-
Only slight dependence on inertial drifts
Change in phase creates ellipticityPolarimetry
consist in studying ellipticity
to extract phase response
60 65 70 75 80 85
0.0
0.2
0.4
0.6
0.8
1.0
Phase [deg.]Inte
nsity
Angle [deg.]
-250
-200
-150
-100
-50
0
50
100
150
200
p- polarization
s- polarization
Intensity
Phase
SPR Polarimetry
MM
Detec
tionDetec
tion LaserLaser
Signal processingSignal processing
Temporal phase modulationSpatial phase modulation
SPR polarimetry
Spatial modulation: Fourier-transform SPR polarimetry
Spatially periodic variation of light polarization
Phase difference shift of inter-
ferometric
fringes
Imaging polarimetry
experimental set-up. Phase measurements by Fast Fourier Transform of interferometric
fringe pattern
Phase resolution = 5*10-2
S. Patskovsky, M. Meunier, A.V. Kabashin, Optics Commun., 281 5492 (2008)S. Patskovsky, R. Jacquemart, M. Meunier, G. De Crescenzo, A.V. Kabashin, Sensors&Act. B, 133 628 (2008)
Temporal modulation: Mechanical modulation method for ultrasensitive phase measurements in SPR
AC Detector Signal at different modulation (+45 and -45 deg.)
Harmonics of the Detector Signal
Mechanical phase modulation by a chopper
Wollaston prism azimuth equal to 45 degrees sets the polarization states of two passing beams at 45 and -45 degrees, respectively, which is equivalent to a 180 degrees phase shift between s and p components
Detection
limit
: Δnmin
= 10-7 RIU
1 2 2 cos( )p s p sAC I I r r ϕ ϕ= − = − 2 2( 1 2) / 2 p sDC I I r r= − = +
S. Patskovsky, M. Maisonneuve, M. Meunier, A. V. Kabashin Opt. Express, 16, 21305 (2008)
Phase resolution = 9*10-3
Temporal modulation: use of photoelastic modulator in SPR polarimetry for wide dynamic range measurements
1st harmonic of modulated intensity:
2nd harmonic of modulated intensity:
3rd harmonic of modulated intensity:
11 2 ( ) cos( )F Ap J M α=
22 2 ( ) sin( )F Ap J M α=
PEM modulation frequency:ϕ
= 42 kHz, sinusoidal
33 2 ( ) cos( )F Ap J M α=
Jn -
Bessel functions; M –
modulation amplitude
1.330 1.332 1.334 1.336 1.338 1.340
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Total
Intensity
Phase
Third
Har
mon
ic
Refractive index
Under certain modulation amplitude (π/2), the 3rd
harmonics signal starts to combine phase sensitivity with wide dynamic range of amplitude measurements
Detection
limit
: Δnmin
= 10-7 RIU, Wide
dynamic
range (up
to 10-2
RIU)
P. P. Markowicz, W.C. Law, A. Baev, P. Prasad, S. Patskovsky, A. V. Kabashin Opt. Express, 15, 1745 (2007)
1 2tan( ) 2 ( ) / 1 ( )F J M F J Mϕ =
Temporal modulation: use of photoelastic modulator in SPR polarimetry for ultrasensitive measurements
11 ( ) cos( )F A J M ϕ=
22 ( ) sin( )F A J M ϕ=
We divide one harmonics by the other one to obtain pure phase response:
42.0 42.2 42.4 42.6 42.80.0
0.2
0.4
0.6
0.8
1.0
Inte
nsity
Angle
100
150
200
250
635nm
Phase [deg.]
670nm
2 3 4 5 6 7 8
0.00
0.02
0.04
0.06
0.08
0.10
2.5% Ar
100% N2
Phas
e [d
eg]
Time [min]
Noise 0.006 deg, Detection limit: 5*10-8 RIU
0 2 4 6 8 10
0.2
0.4
0.6
0.8
1.0
48nm
35nm
25nm
Angle
Inte
nsity
Calibration point
150
200
250
300
350
400
450
Phase
Intensity and phase measurement in the same time
Medium = air
48 nm
35 nm
25 nm
S. Patskovsky, M. Vallieres, M. Maisonneuve, In-Hyouk Song, M. Meunier, A. V. Kabashin Opt. Express, 17, 2255 (2009)
Designing efficient zero-point using phase properties of light
Phase and intensity responses related to antibody binding to surface immobilized lysozyme
Examples of applications of phase-sensitive SPR
0 2 4 6 8 10 12
0.00
0.05
0.10
0.15
0.20
Thiolated BSA + Streptavidin-Maleimide(1.3 nM)
Thiolated BSA + Streptavidin-Maleimide(13 nM)
Thiolated BSA + Streptavidin-Maleimide(0.13 m M)
Rel
ativ
e si
gnal
cha
nge
(3rd
har
mon
ic)
Time (min)
Response curves when samples of Streptavidin-Malemide/Thiolated
BSA complex at various concentrations were injected into the sensor head
W-C. Law, P. Markowicz, K-T. Yong, I. Roy, A. Baev, A. V. Kabashin, H-P. Ho, P. N. Prasad, Biosens& Bioelectron., 23, 627-632 (2007)
S. Patskovsky, R. Jacquemart, M. Meunier, G. De Crescenzo, A.V. Kabashin, Sensors&Act. B, 133 628 (2008)
Why phase sensitive schemes have lower detection limit compared to amplitude-based ones???
1. The change of electric field is much bigger in the SPR dip, where main change of phase and not amplitude takes place
2. In most schemes of SPR measurements the detector-related shot noise is lower than other noises such as the noise associated with the light source. On the other hand, light sources normally have much better stability of phase characteristics compared to amplitude ones
60 65 70 75 80 85
0.0
0.2
0.4
0.6
0.8
1.0
Phase [deg.]
Inte
nsity
Angle [deg.]
-250
-200
-150
-100
-50
0
50
100
150
200
p- polarization
s- polarization
Intensity
Phase
3. Phase offers much more efficient and flexible methods for averaging, image treatment etc., which gives additional tools to improve the sensor sensitivity
/ϕ ϕΔ ≈10-6Phase noise: /I Iχ = Δ ≈10-2Relative intensity noise of common lasers :
Commercial implementation of SPRI: Cambridge Consultants Ltd, UK
Commercial implementation of SPRI: Alphasniffer Inc.
JILA-NIST-CUWorkshop on
Interferometric/Surface Plasmon Resonance (SPR) Array Biosensors
JILA Main Auditoriumhttp://jilawww.colorado.edu/news/hallworkshop.html
http://www.alphasniffer.com/data/PR/JILA%20Workshop%20Agenda.pdf440 UCB
University of ColoradoBoulder, Colorado, USA
April 18, 20061-5 PM
OrganizerProfessor John Hall, JILA, 2005 Physics Nobel Laureate
Sub-project II: Miniaturization and cost
reduction
Miniaturization of SPR biosensors
Waveguide-based SPR Biosensors
Concept of Si- based SPR
Current methods of SPR miniaturization: waveguide-based coupling
1. Plasmon is coupled from a single mode waveguide (Wilkinson, 1995)
Disadvantages:-
Coupling takes place at effective grazing angles, drastically decreasing the sensitivity of SPR sensing transduction-
Coupling takes place at relatively short wavelengths (green-red)
Multi-mode waveguide
Photonics crystal waveguide-based Surface Plasmon Resonance biosensor
1. M. Skorobogatiy, A.V. Kabashin Opt. Express., 14, 8419-8424 (2006)2. M. Skorobogatiy, A.V. Kabashin Appl. Phys. Lett., 89, 143518-143521 (2006)3. E. Pone, A. Hassani, A. Kabashin, M. Skorobogatiy, Opt. Express, 15, 10231 (2007) 4. B. Gauvreau, A. Hassani, M. Fassi Fehri, A. V. Kabashin, M. Skorobogatiy, Opt. Express, 15, 11413 (2007)
Instead of single mode waveguide, we propose to couple plasmon from a Gaussian-like leaky mode of a single mode photonics crystal waveguide
-
Light confinement in the low refractive index core is achieved by a surrounding multi-layer reflector-
Coupling efficiency for Gaussian mode is high due to good spatial mode matching-
Coupling to PCW can be simplified by choosing core size larger than the pumping wavelength
Photonics crystal waveguide
Effective index of the mode can be less than that of the core
Phase matching can be obtained in an arbitrary pointby an appropriate PCW design
Concept of photon crystal waveguide (PCW)-based SPR biosensor
M. Skorobogatiy, A.V. Kabashin Appl. Phys. Lett., 89, 143518-143521 (2006)
Sensitivity of PCW-SPR biosensor with various thicknesses of gold film
Sensitive response of photonics crystal waveguide-based SPR
Sx energy flux strongly depends on the refractive index of ambience
Plasmonics: Sensors tune in, Nature Photonics, 19 October 2006
Phase-sensitive Bragg-grating waveguide-based SPR
G. Nemova, A. V. Kabashin, R. Kashyap, J. Am. Opt. Soc. B., 25, 1673-1677 (2008)
Plasmon is coupled from a Bragg grating structure formed on a waveguide
Si-based SPR
Commercial SPR systems:-Bulky-Expensive
(200 k$ et plus)-Laboratory
applications only
S. Patskovsky, A. V. Kabashin, M. Meunier, J. Luong, Sens&Act. B, 97 409 (2004)
Miniaturization
Limitation: glass-based
technology
Solution: Adaptation of SPR-based technology to Si
Miniaturization: Si-based microfabrication methods are well
developed
Possibly, properties of Si and especially its relatively high refractive index (3.45) and transparency in IR can provide some advantages for biosensing
Si λ=1100-10000 nm
KSP
Gold
Miniaturization and cost reduction: Si-based phase-sensitive SPR
500 1000 1500 2000 250045
50
55
60
65
70
75
80
Wavelength [nm]
SPR
Ang
le [d
eg.]
21.8
21.9
22.0
22.1
22.2
22.3
22.4
22.5
Glass
Silicon
1. S. Patskovsky, A. V. Kabashin, M. Meunier, J. Luong, JOSA A. 20, 1644 (2003)2. S. Patskovsky, A. V. Kabashin, M. Meunier Sens&Act. B, 97 409 (2004) 3. S. Patskovsky, A. V. Kabashin, M. Meunier, J. Luong, Appl. Opt. 42, 6905 (2003) 4. S. Patskovsky, A. V. Kabashin, M. Meunier Anal. Lett., 36, 3237 (2003)5. S. Patskovsky, A. V. Kabashin, M. Meunier Opt. Mater., 27, 1093 (2005)
1.32 1.34 1.36 1.38500
1000
1500
2000
Glass
Silicon
Wav
elen
gth
[nm
]
Bulk refractive index
Si-based phase-sensitive SPR micro-sensor
Cover plateMicrochannels
Optical fibres
Microfabrication of Si- based SPR biosensor
Si-based total internal reflection phase-sensitive sensor
Phase shifts that p-
and s-polarized light experience in total internal
reflection (TIR)2 2 1/ 2( sin 1)2arctansin tanp s
nn
ϕδ δϕ ϕ
−Δ = − =
20 40 60 80 1000
10
20
30
40
50
60
70
80
90
100
ΔSF11
ΔSi = δp − δs
ΔBK7
nm=1.4
Water
TIR
Pha
se D
iffer
ence
[deg
.]
Angle [deg.]
Differential phase shifts for the three prism materials: Silicon; glass SF11 and BK7
Silicon based TIR phase-sensitive sensor – more sensitive than glass-based TIRSimple system – high intensity of TIR light
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
0
4000
8000
12000
16000
SF11
BK7
Silicon
Sens
itivi
ty d
Δ/d
n m [d
eg.]
Refractive index nm
RI sensitivity calculated at phase difference Δ
equal 10 degree
S. Patskovsky, M. Meunier, A.V. Kabashin Opt. Express, 15, 12523-12528 (2007)
The detection limit approaches 10-5 RIU
20 22 24 26 280
20
40
60
80
Δn = 3.1*10-3Δn = 9.3*10-3
Si prism
Phas
e di
ffere
nce
[deg
.]
Angles [deg.]
0 5 10 15
62
64
66
68
70
72
8%
2.7%
1%0.4%
Phas
e di
ffere
nce
[deg
.]
Time [min]
0 2 4 6 8 10
0
2
4
6
88%
2.7%
1%0.4%
Phas
e [d
eg.]
Refractive index x10-3
Real time differential phase measurements of glycerine-water mixtures with various weight ratios
Si-based total internal reflection phase-sensitive sensor
S. Patskovsky, M. Meunier, A.V. Kabashin Opt. Express, 15, 12523 (2007)
Sub-project III: Development of novel
nanoplasmonics architectures
The
SPR response
can
be
amplified
by the
use of
gold nanoparticle
markers linked
to biospecies
(L. Lyon et.al.
Anal. Chem., 1998)
SPR biosensors with nanoparticle-based signal amplification
Changes of
index of
refraction
can
be
too
small
under
tests with
drugs, vitamins
and
some
proteins
What we propose
Markers of
different
materialsPhase sensitivityResponse
in near-IR
(1100-2300 nm)
SPR biosensors with nanoparticle-based signal amplification
-2 0 2 4 6 8 10 12 14 16 18 20
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
Avidin (5ng/ml)
PBS
PBS
Rel
ativ
e S
igna
l Cha
nge
Time (min)
SPR signal0 5 10 15 20 25 30
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030Avidin with
Au NP (50pg/ml)
Water
WaterRel
ativ
e si
gnal
cha
nge
Time (min)
The
nanoparticles
can
amplify
the
signal by 100 and
more!!!
SPR amplified
by nanoparticles
Al
TiPt
PdAg
AuCu
Pha
se R
espo
nse
(Arb
. uni
ts)
Nanoparticle Material
What
type of
nanoparticle
material
generates
the
strongest
SPR response???
S. Patskovsky, A. V. Kabashin, M. Meunier Opt. Mat., 27, 1093 (2005)
v1_s8_60
X Data
400 500 600 700 800
Psi
0
5
10
15
20
25
30
35
Pha
se
0
100
200
300
400
500
PsiPhase
Phase sensitivity
V. Kravets, F. Schedin, A. V. Kabashin, A. N. Grigorenko Appl. Phys. Lett., submitted
Double gold nanopillar array- based sensor
Use of new nanoplasmonics architectures on the surface
Conclusion
Plasmonics
offers unique tools for the detection and studies of biological binding/recognition events on gold surface
We achieved significant progress in the developing of plasmonics
biosensing
methods with: (a) improved
sensitivity; (b) miniatuarized
designs; (c) novel nanoscale
architectures