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.

Introduction

Surface plasmon fluorescence immunoassays

Bridge SPR technology and nano-world

Development of surface plasmon diffraction sensor

Summary

Outline

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?

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)

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

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

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…

: 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

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

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

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

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.

-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

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.

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)

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

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

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

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

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

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

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

Diffraction patterns

Surface plasmon microscopy images

Diffraction photographs

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

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.

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.

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

-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

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.

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

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)