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