SURFACE PLASMON RESONANCE (SPR) BIOSENSORS: … · 2010. 11. 30. · PLASMONIC ARRAY BIOSENSORS SPR image corresponding to 2×1 mm2 area. APPROACH: • Arrayof~100sensingchannels

1

SURFACE PLASMON RESONANCE (SPR) BIOSENSORS: ADVANCES,

JIŘÍ HOMOLA

CHALLENGES AND APPLICATIONS

INSTITUTE OF PHOTONICS AND ELECTRONICSACADEMY OF SCIENCES OF THE CZECH REPUBLIC

FJFI Seminar November2010

Established in 1954.

Over 150 staff members in three Research divisions and Support

INSTITUTE OF PHOTONICS AND ELECTRONICS (UFE)

units.

Funding: 60% governmental support, 40% grants & contracts.

Institute Mission

Institute of Photonics and Electronics, Prague

Basic and applied research in three Thrust Areas:Area I: Photonics

Area II: Materials for optoelectronics

Area III: Signals and systems

2

Department of Optical

Sensors

Laboratory of Optical

Fibers

Department of Guided-Wave

Photonics

DIVISION OF PHOTONICS

SensorsFibersPhotonics


BiophotonicsOptical Communications

Analytics

BIOSENSORS AND WHY THEY ARE NEEDED


3

ANALYTICAL NEEDS TODAY AND TOMORROW

Environmental monitoring

f

Technologies for rapid and sensitive detection of biolo-gical species are needed in numerous important sectors.

Food safety

Life sciences and pharmaceutical research

Medical diagnostics


Health care at home Networks of sensors

Personalized health care

CURRENT AND EMERGING BIOANALYTICAL TECHNOLOGIES

Limited to central laboratories Require trained personnel Time consuming

Conventional methods

Suitable for bioanalytical

Time consuming

HIGH COST,LOW SPEED OF ANALYSIS

Biosensors


yapplications in the field

Enable permanent deployment and unattended operation

4

Biosensors are devices consisting of a biomolecularrecognition element and a sensor hardware,

AFFINITY BIOSENSORS

Biomolecular

Target analyte

Non-target substances}Antibodies

ProteinsDNA RNA

MechanicalElectricalMagnetic


which translate the binding event between the target molecules and biorecognition element into an output signal.

Biomolecularrecognition

element

DNA, RNAPeptidesMIPs

MagneticOptical

OPTICAL AFFINITY BIOSENSORS

OPTICAL AFFINITY BIOSENSORS are devices consisting of a biorecognition element and optical hardware, which translates the binding event between the target molecule (analyte) and biorecognition element into an output signal(analyte) and biorecognition element into an output signal.


Antibodies, nucleic acids (DNA, RNA), peptides, polymers with molecular imprint (MIP), etc.

Biorecognition elements – examples:

5

Measure fluorescence

MAIN TYPES OF OPTICAL AFFINITY BIOSENSORS

1. Label-based affinity biosensors• Sensors based on fluorescence spectroscopy.

Measure fluorescence

• Interferometric sensors (Mach-Zehnder integrated optical interferometer, white light interferometer).

2. Label-free affinity biosensors


Measure refractive index

• Sensors based on spectroscopy of guided waves (grating coupler, resonant mirror, surface plasmon resonance sensor).

DNA hybridization DNA Chip

FLUORESCENCE-BASED AFFINITY BIOSENSORS

Antibody-antigen assay


GenePix, Molecular Devices Corp., USA

Portable biosensor, Constellation

Technology (USA).

6

CONCEPT OF LABEL-FREE OPTICAL AFFINITY BIOSENSORS

tive

in

de

xre

sp

on

se)

dnRI

Binding event,

Biorecognition

Analyte

Time

Re

frac

t(S

en

so

r

Binding of analyte tobiorecognition element

voldR

cI

c Change of refractive index, n


Optical wave

Transducer

element p

Change of optical wave parameter, p

p

n2

n1

Ref

lec

tiv

ity

Analyte

Wavelength [nm]

SURFACE PLASMONS AND SPR BIOSENSORS


SPR Metal

Substrate

Light Beam

7

LOCALIZED SURFACE PLASMONS (LSP)

I. Nanoparticles CHARACTERISTICS OF LSP:

PENETRATION DEPTH, LField extent: Ldiel = 10 - 40 nm

II. Arrays of nanoobjects

Figure of merit (FOM):FOM = 0.5 – 10


[1] M. A. Otte et al. , ACS NANO 4, 349–357 (2010).[2] J. N. Anker et al., Journal of Physical Chemistry C, 113, 5891-5894 (2009).[3] Jonsson et al, Nano Letters, 7 (2007).

SEM image: Gold nanorods 1 Nanopyramid array 2 Nanohole array 3

CHARACTERISTICS OF PSP:METAL DIELECTRIC

(Vacuum)

PROPAGATING SURFACE PLASMONS (PSP)

Propagation constant:

m dε εωβ

SURFACEPLASMON

Field extent: Ldiel = 150 - 400 nm

Figure of merit (FOM):FOM = 60 – 120

m d

m d

β=c ε +ε


A propagating surface plasmon at a metal–dielectric interface.

Figure of merit for surfacechanges(sFOM):

Comparable to LSP

8

0.4

0.6

0.8

1.0

Ref

lec

tivi

ty

BIOSENSORS BASED ON SPECTROSCOPY OF PROPAGATING SURFACE PLASMONS

600 700 800 9000.0

0.2

Wavelength [nm]

expE i z t

zk


Binding-induced propag. constant change:Matching condition:

2sin Re{ }z pnk

vol

dn

dK

c

Kretschmann geometry of the ATR method.

SURFACE PLASMON RESONANCE SENSOR PLATFORMS


9

Sensors based on angularspectroscopy of surfaceplasmons: BIAcore S51 (left), BIAcore 3000 (middle) Spreeta

COMMERCIAL SENSORS BASED ON SPECTROSCOPY OF SURFACE PLASMONS

BIAcore 3000 (middle), Spreeta sensor, TI (right) .

FEATURES:• High resolution (~10-7 RIU )LIMITATIONS:• Costly (over 300kEUR)


• Temperature controlled

• Multiple sensing channels

Applications: biomolecular interaction analysis

• Bulky (80 kg)• Requires trained personnel• Designed for use in the lab

LABORATORY SPECTROSCOPIC SPR SENSOR PLASMON IV Spectroscopy of surface

plasmons.

Four sensing channels, 2

Channel 1

n=1.33

n=1.32

Lig

ht

Inte

nsi

ty

Channel 2

n=1.33

n=1.32

Polychromaticradiation

Prism

Ch l A Ch l B

SP 1 SP 2

Metal layerSampleg

(flow chamber volume 0.5 μL per channel)

Temp. stabilization (stability < 0.02°C)

RI RESOLUTION4

1

3

600 650 700 750 800 850 900 950

Wavelength [nm]

Channel A Channel B


1. sensor head 2. spectrometer3. light source 4. peristaltic pump

RI RESOLUTION:< 2×10-7 RIU

OPERATING RANGE:1.32-1.45 RIU

1

10

PORTABLE WDMSPR SENSOR

n=1.33n=1.32

Lig

ht

Inte

nsi

ty

Polychromaticradiation Prism coupler

Metal layer

600 650 700 750 800 850 900 950

n 1.32

Wavelength [nm]Channel 1 Channel 2

SPW1 SPW2Sample


A portable 8-channel SPRWDM sensor for field use. RI RESOLUTION:4x10-7 RIU

J. Homola, et al., Electronics Letters 35, 1105-1106 (1999).P. Adam, J. Dostálek, J. Homola, Sensors and Actuators B 113, 774-781 (2006).

SPECTROSCOPY OF SURFACE PLASMONS ON DIFFRACTIVE STRUCTURESSurface plasmon resonance coupler and disperser (SPRCD) simultaneous-ly excites a surface plasmon via 2nd order of diffraction and disperses light diffracted into the 1st diffraction order over a position sensitive detector.

1.2

TE

)

ADVANTAGES

Collimatedbeam of

polychromaticlight

Surfaceplasmon

Detector

1st diffraction

820 830 840 850 860 870 880 8900.0

0.2

0.4

0.6

0.8

1.0

n=1.34n=1.32

1st o

rder

eff

icie

ncy

(T

M/T

Wavelength [nm]


SPRCD: principle of operation

ADVANTAGES: Low-cost (no spectrometer) Compact Chips compatible with mass

production

st d act oorder

SPRCD

O. Telezhnikova, J. Homola, Optics Letters, 31, 3339-3341 (2006).

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COMPACT SPRCD SENSOR FOR FIELD USE

SENSITIVITY:615 nm/RIU

RESOLUTION:< 3×10-7 RIU


< 3×10 RIU

OPERATING RANGE:1.33-1.35 RIU

Laboratory prototype of 6-channel SPRCD sensor.

M. Piliarik, M.Vala, I. Tichý, J. Homola, Biosensors & Bioelectronics, 24 3430–3435 (2009).

Parallelmonochromatic

2D arraydetector

P l i

RI RESOLUTION:3×10-5 RIU *

TYPICAL PERFORMANCE:

SPR IMAGING: PRINCIPLE OF OPERATION AND PERFORMANCE

beam

G ld d

Polarizer

Array of sensingspots

CouplingPrism

Imagingoptics

DETECTION OF NUCL. ACIDS:10NM (18-MER)**

DETECTION OF PROTEINS:1NM (ANTI-FLAG)***

CHALLENGES:


Gold-coated sensor surface

p

Sample

* Fu, E., et al., Review of Scientific Instruments, 75, 2300 - 2304 (2004).** Lee, H.J., T.T. Goodrich, and R.M. Corn, Analytical Chemistry, 73, 5525 - 5531 (2001).*** Wegner, G.J., H.J. Lee, and R.M. Corn, Analytical Chemistry, 74, 5161 - 5168 (2002).

C G S: Operating range. Noise due to

light level fluctuations. Image contrast.

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SPR IMAGING WITH POLARIZATION CONSTRAST FOR HIGH-THROUGHPUT SCREENING

Parallelmonochromatic

2D arraydetector

Li ht tmonochromaticbeam

SPR sensor chip

Polarizers

SPR active spot

/4 Waveplate

Imagingoptics

Sample

CouplingPrism

Illuminatedarea

Surface plasmon

Gold layer

Light spot

Sensor chip

TE/TM retardingtitanium layer

Al O spacer2 3

Pair of sensing spots


SPR imaging with polarization contrast and optical multilayer, system layout and detail of the SPR sensor chip.

M. Piliarik, H. Vaisocherova, J. Homola, Biosensors & Bioelectronics, 20, 2104 - 2110 (2005).

Totally reflected lightIncident lightGlass substrate


1.301.321 34

2

3

4

ma

lize

d si

gnal

Image of the SPR sensing chip for rows

1.341.361.381.401.421.45

1.30 1.35 1.400

10

20

Spot type I Spot type II

Rel

ativ

e in

tens

ity [%

]

Refractive index [RIU]

Optimum operating range

0

1

No

rm


of sensing spots exposed to liquids of different refractive indices.

M. Piliarik, H. Vaisocherová, J. Homola, Sens. Act. B, 121. 187 (2007).M. Piliarik, H. Vaisocherová, J. Homola, Biosen. Bioel., 20, 2104 (2005).

RI RESOLUTION:1x10-6 RIU

Refractive index [RIU]

Light intensity vs. refractive index. Structure: Ti layer, Al2O3 layer (200 nm), and SPR-active Au layer (40 nm).

13


WaveplatePolarizerPolarizer

C) A)

B)

CC

D

Prism

Flow cellGold layer

Mirror 1

SPRchip {

A)

B) C)

D


M. Piliarik, J. Homola, Sensors and Actuators B, 134, 353–355 (2008).J. Ladd, T. Allen, M. Piliarik, J. Homola, S. Jiang, Analytical Chemistry 80, 4231–4236 (2008).M. Piliarik, L. Párová, J. Homola, Biosensors & Bioelectronics, 24, 1399-1404 (2009).

Gold layerMirror 2

High-throughput SPR imaging sensor with polarization control.

RI RESOLUTION:2x10-7 RIU

BIORECOGNITION ELEMENTS AND THEIR IMMOBILIZATION


14

BIOMOLECULAR RECOGNITION ELEMENTS AND THEIR IMMOBILIZATION

Main requirements:

Choice of immobilization method depends on the type of biorecogntion element and specifics of application.

1. High, controlled density of biorecognition elements

2. Non-fouling background

1. Covalent couplingMain approaches:


p g2. Hydrophobic interaction3. Electrostatic interactions4. Affinity interactions

(e.g. streptavidin – biotin)

FUNCTIONALIZATION OF SPR SENSORS USINGSTREPTAVIDIN – BIOTIN CHEMISTRY

SubstrateSubstrate


SubstrateGold layerSubstrate Substrate

Gold layerSubstrate

15

SPATIALLY-RESOLVED FUNCTIONALIZATION FOR PLASMONIC ARRAY BIOSENSORS

SPR image corresponding to 2×1 mm2 area.

APPROACH:

• Array of ~ 100sensing channels

Sensor surface

Sensor surface

flow

FEATURES:


• Array of ~ 100sensing channels • Sensing channel dimensions: 150 x

150 μm• Accessible DNA probes: (3-8) x 1012

DNA/cm2

M. Piliarik, H. Vaisocherová, J. Homola, Sensors and Actuators B, 121, 187-193 (2007).

APPLICATIONS OF SPR BIOSENSORS


16

1. Investigation of molecules & their interactionsReal-time study of molecular interactions allowing determination of specificity, interaction models, ki ti t ilib i t t th d i

SPR BIOSENSORS: MAIN APPLICATION AREAS

kinetic rates, equilibrium constants, thermodynamic constants, and epitope mapping.

2. Detection, identification and quantification of chemical and biological substances.• Food safety (foodborne pathogens and toxins)


J. Homola, Chemical Reviews, 108, 462-493 (2008).

• Medical diagnostics (disease biomarkers, antibodies)• Environmental monitoring (endocrine disrupting

compounds)

SCREENING OF OLIGONUCLEOTIDES WITH MODIFIED LINKAGE FOR ANTISENSE THERAPY

29 oligonucleotides (dT15) with different structural modifications at

Sensor response to binding of oligonucleotides with selected modified linkages to natural rA23A B

0

1

2

3

4

buffer

buffer

d[(A)14

T]

d[(MeOEtO

T)14

T] (#28)S

ens

or

resp

onse

[nm

]

bufferbuffer

d[(B)14

T]

d[ara(Tpc)14

T] (#9)

concentration of 100nM interacted with bound to rA23 on the sensor surface.

A B


110 120 130 140 110 120 130 140

Time [min]

• Rapid screening (YES or NO in 15 min)

• Small sample consumption (0.1-100nM)

• Monitoring kinetics of the oligonucleotide interactions

17

SCREENING OF OLIGONUCLEOTIDES WITH MODIFIED LINKAGE FOR ANTISENSE THERAPY

0

1

2

3

4

4

1.

2. 3. 4. 5.

7. 8. 9. 10. 11. 12.

6.

0

1

2

3

4

0

1

2

3

4

2

3

4

13.

Sen

sor

resp

on

se [

nm

]

14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24.

Natural dT15


0

1

100 120 140

0

1

2

3

4

100 120 140 100 120 140 100 120 140 100 120 140 100 120 140

25.

26.

27.

Time [min]

28. 29.

30.

KINETIC ANALYSIS OF OLIGONUCLEOTIDES WITH MODIFIED LINKAGE FOR ANTISENSE THERAPY

Duplex NA0.6

0.8 II. d[(X)14

T]

0.6

0.8 I. d[T(TX)7] (S)

PO

THO

OOH

(S)-

se [

nm

]

Oligo #

kaD

[M-1s-1]kdD

[s-1]kaT

[M1s-1]kdT

[s-1]KdD

[nM]KdT

[μM]

p

Triplex NA

0 5 10 15 20 25 30

0.0

0.2

0.4

O

PO T

HO

OO

OCH3

Time [min]

0 10 20 30 40 50

0.0

0.2

0.4 O

Sen

sor

resp

on

s

Time [min]


0 5 10 15 20 25 30

0.00

0.04

0.08

0.12 III. dT15

Se

nso

r re

spo

nse

[n

m]

Time [min]

# [M s ] [s ] [M s ] [s ] [nM] [μM]

I 4.96e5 3.16e-3 2e3 5.7e-5 6.4 0.03

II 4.76e5 7.21e-3 2.87e3 1.54e-4 15.2 0.05

III 7.05e5 7.94e-2 4.09e2 1e-4 112.7 0.24

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SPR FOR ENVIRONMENTAL MONITORING:DETECTION OF ENDOCRINE DISRUPTORS

I. INCUBATION

Antibody

Anal te

1.0

1.2

nse

0 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml

II. DETECTION OF FREE ANTIBODY

Analyte

0 5 10 15 20 25

0.0

0.2

0.4

0.6

0.8

Se

ns

or

resp

on


Inhibition assay detection format.

BSA-EDC conjugate

Gold layerSubstrate

Time [min]

Detection of atrazine using inhibition assay. Kinetic response

to unreacted antibody.

SPR FOR ENVIRONMENTAL MONITORING:DETECTION OF ENDOCRINE DISRUPTORS

0.8

1.0

se [

a.u

.]

LODS:ATR – 70 pg/ml

10-3 10-2 10-1 100 101 102 1030.0

0.2

0.4

0.6

atrazinebenzo[a]pyrene4-nonylphenol2,4-dichlorophenoxyacetic acid

Se

ns

or

resp

on

s


Calibration curves for detection of atrazine (ATR), 2,4-dichlorophenoxyacetic acid (24D), 4-

nonylphenol (4NP) and benzo[a]pyrene (BaP).

BaP – 50 pg/ml

4NP – 260 pg/ml

2,4-D – 160 pg/ml

J. Dostálek, J. Přibyl, J. Homola, P. Skládal, Anal. and Bioanal. Chem., 389, 1841–1847 (2007).

10 3 10 2 10 1 100 101 102 103

Analyte concentration [ng mL-1]

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SPR FOR FOOD SAFETY:DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

I. DIRECT DETECTION

3

4

5

BSAift

[nm

]

II. AMPLIFICATION

0 20 40 60 80

-1

0

1

2

3

Amplification(antibody capture)

Direct detection(target capture)

a-SEB

BSASEB

BSA

SP

R w

avel

eng

th s

hi


SPR sensor response to SEB. (SEB concentration - 25 ng/ml, a-SEB

concentration – 3 g/ml).Sandwich detection format.

J. Homola et al., International Journal of Food Microbiology, 75, 61-69 (2002).

0 20 40 60 80

Time [min]

10

12 Amplified response in milk

Dire

e [n

m]

1.0

1.2

Direct response

Amplified response

SPR FOR FOOD SAFETY:DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN BCalibrationcurve:

0

2

4

6

8

ect respo

nse [n

m]

Am

plif

ied

res

po

nse

0.0

0.2

0.4

0.6

0.8 Direct response


1 10

0

SEB concentration [ng/ml]

0 0

SEB in buffer and milk (sandwich format): LOD: 0.5 ng/ml

Botulinum neurotoxins in buffer, honey (20%): LOD: <1 ng/mlJ. Ladd, et al., Sensors & Actuators B, 130, 129-134 (2008).

20

SPR FOR MEDICAL DIAGNOSTICS:DETECTION OF ANTIBODY AGAINST EB VIRUS

2.5

3.0

3.5

e [n

m]

30m

M

30m

M

M0m

M

2000

ng

/ml

00 n

g/m

l

0 n

g/m

l

2 n

g/m

l

ng

/ml

180 210 240 270 300 330

-0.5

0.0

0.5

1.0

1.5

2.0

Sen

sor

resp

on

se

Na

OH

3

Na

OH

NaO

H 3

0m

M

NaO

H 3

0

NaO

H 3

0m

M

anti

-EB

NA

2

anti

-EB

NA

20

anti

-EB

NA

20

anti

-EB

NA

2

anti

-EB

NA

0.

2

bu

ffer

bu

ffer

bu

ffer

bu

ffer

bu

ffer

bu

ffer

bu

fferbu

ffer

bu

ffer

bu

ffer

bu

ffer

0 20 40 60 80

0

2

4

6

BSA-CMV (reference surface)

BSA-EBNA-1

Glutaraldehyde0.5%

BSA-EBNA-1BSA-CMV50 µg/ml

PBS

PBS

PBS

Sen

sor

resp

on

se [

nm

]


SPR sensor response to increasing concentration of anti-EBNA using regenerated peptide surface.

Time [min]

Monitoring immobilization of BSA-EBNA peptide

conjugate on gold.

0 20 40 60 80Time [min]

H. Vaisocherová, K. Mrkvová, M. Piliarik, P. Jinoch, M. Šteinbachová, J. Homola, Biosensors and Bioelectronics 22, 1020 (2007).

2.5

3.0

e [n

m]

anti-EBNA-1 in 10 mM PBS, 0.5% BSA anti-EBNA-1 in 10 mM PBS,

0.5% BSA, 1% human serum

SPR FOR MEDICAL DIAGNOSTICS:DETECTION OF ANTIBODY AGAINST EB VIRUS

0.0

0.5

1.0

1.5

2.0

Sen

sor

res

po

nse

LOD:0.2 ng/ml

VARIABILITY:< 15 per cent


Sensor response to anti-EBNA in buffer (14 binding experiments for each concentration) and in serum.

0.1 1 10 100 1000Concentration of anti-EBNA-1 [ng/ml]

< 15 per cent

H. Vaisocherová, K. Mrkvová, M. Piliarik, P. Jinoch, M. Šteinbachová, J. Homola, Biosensors and Bioelectronics 22, 1020 (2007).

21

SPR FOR FOOD SAFETY:DETECTION OF BACTERIAL PATHOGENS

2 5

3.0

3.5

4.0

shift

[nm

] PBS Apple Juice pH 3.7 Apple Juice pH 7.4

LOD:

0.0

0.5

1.0

1.5

2.0

2.5

104 5 104 105 106 107

SP

R w

avel

engt

h s LOD:

E. coli:104 cell/ml

C. jejuni: 5×104 cell/ml

S. typhimurium:


104 5x104 105 106 107

Cell concentration [cfu/ml]

SPR sensor response to different concentration of E. coli in buffer and in apple juice

(sandwich detection format).A. D. Taylor, Q. Yu, S. Chen, J. Homola, S. Jiang, Biosensors & Bioelectronics 22, 752 (2006).

yp5×104 cell/ml

L. monocytogenes: 104 cell/ml

SUMMARY

Surface plasmon resonance biosensors present one of the most advanced label-free optical biosensor technologies.

Various SPR sensor platforms and functionalization

Enable rapid detection with limits of detection for small and medium-size analytes 10 pg/ml - 100 ng/ml levels (without amplification).

There are numerous prospective applications in

Various SPR sensor platforms and functionalizationchemistries are available to meet needs of specific applications.


There are numerous prospective applications in important sectors such as medical diagnostics, environmental monitoring, food safety and security.

New technologies need to be competitive on the basis of factors such as: costs, robustness, user-friendliness, sensitivity, and specificity.

22

ACKNOWLEDGEMENTS

Collaborators:University of Washington, Seattle, USA (S. Jiang, Q. Yu)Institute of Macromolecular Chemistry ASCR, Prague (E. Brynda)Institute of Hematology and Blood Transfusion Prague (J E Dyr)

Sponsors:National Science Foundation of CRAcademy of Sciences of CR

Institute of Hematology and Blood Transfusion, Prague (J. E. Dyr)Faculty of Mathematics & Physics, Charles University (J. Štěpánek)Institute of Organic Chemistry & Biochemistry ASCR (I. Rosenberg)


Academy of Sciences of CRMinistry of Health of CRUS Food and Drug AdministrationEuropean CommissionPhenogenomics, Inc. (USA)

Documents

SURFACE PLASMON RESONANCE (SPR) BIOSENSORS: … · 2010. 11. 30. · PLASMONIC ARRAY BIOSENSORS SPR image corresponding to 2×1 mm2 area. APPROACH: • Arrayof~100sensingchannels