More information with SPRimaging:More information with SPRimaging:More information with SPRimaging:More information with SPRimaging:

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A typical SPR imaging experiment

SDF Chemokine recognition on an

oligosaccharide chip

Time (s)C

ha

ng

e i

n r

efl

ec

tivit

y (

%)

sensorgrams

regeneration

dissociation

association

Information: affinity data

How to get more information from

one SPR imaging experiment ?

Molecular interaction measurement

use a versatil chemistry

increase the number of parameters

increase the discrimination of the recognition step

Imaging of complex systems:

lipid layers

cells

Initially developped for the MICAM DNA chips :

microelectrode array ODN array

CTTGT

CT CGT

CTGGT

via pyrrole/ ODNpyrrole copolymerization

+

H

N

Oligonucleotide

N

H

N

Oligonucleotide

N

H

N

Electro-oxidization

Nucleic Acids Res, 1994, 22: 2915

Anal Biochem, 1998, 255: 18

Biosensors&Bioelectronics, 1998, 13: 629

Mikrochim Acta, 1999, 131: 3

Clin. Chem, 2001, 47(2):194

Micam Chip

Fluorescence detection

on a Micam Chip

Chemistry for DNA chip:

example of polypyrrole based grafting process

O

N

N

OH

O

D MTO

C H3

N H (C H2)6N H2

O

N

N

OH

O

D MTO

C H3

N H ( C H2)6N H

O

N

NO

D MTO

C H3

O

PN C (C H2)2O N [ C H (C H3)2]2

C O(C H2)5 N

N H (C H2)6N H C O(C H2)5 N

O

N

NO

H O

C H3

O

P

N H ( C H2)6N H C O( C H2)5 N

O O-

O

NHS Caproyl pyrrole

Pyrrole-phosphoramiditebuilding block

OLIGONUCLEOTIDE

Pyrrole-ODN

Oligonucleotidesynthesis

Bis(diisopropylamino)cyanoethoxyphosphine

Pyrrole phosphoramidite

polypyrrole : intelligent glue for biological applications

BIOLOGY OPTICS

(applications) (detection)

CHEMISTRY

protein

ssDNA

peptide

phosphoramidite

pyrrole

Biotin-pyrrolepyrrole tool-box

double stranted DNA

Chemical versatility :

Grafting of various biomolécules

NHS pyrrole

oligosaccharides

N

Gold layer

Glass

For SPR support

Gold : used for SH- grafting (not so easy !)

used as a working electrode , OK for polypyrrole electrochemistry

Addressing : only one electrode !

BUT HOW TO ADDRESS THE ELECTROCHEMICAL REACTIONS ?

For Micam (addressed µelectrodes)

the chip is dipped into the solutions

and one µelectrode is activated

polypyrrole chemistry applied to SPR substrates

Szunerits et al Langmuir (2004)

First strategy :

SECM-like approach monitored by SPR imaging

writing and simultaneous reading of micropatterns

REsolution

Microelectrode

working electrode

Polypyrrole

spots

Light beam (illumination)Light beam detection

CCD camera

CE

REsolution

Microelectrode

working electrode

Polypyrrole

spots

Light beam (illumination)Light beam detection

CCD camera

CE

SECM based pyrrole polymerization coupled with

Real time monitoring of the polymer synthesis by SPRi

Change the solution for each ODN to graft

Wash the cell to avoid cross contamination

Tool for electrosynthesis monitoring

D

A

B

C

polypyrrole spots

C E W E

x

yz

potentiostat

Cell volume : Cell volume : 55µµLL

Covalent Covalent graftinggrafting in 0.5sec, in 0.5sec, ddp 2Vddp 2V

spot : spot : 400400µµmm φφ, 5 nm , 5 nm thickthick

20 spots/chip20 spots/chip

Pyrrole 20mM

Phosphate buf. 0.1M

ODN-pyrrole 1µM

patent wo 00/34227

Synthetic metals, 2001, 121: 1443

stainless steel electrochemical pin

Second strategy:

liquid confining in a mobile electrochemical cellfirst generation using a pipette tip including a Pt wire: 20spots/chip

D

A

B

C

polypyrrole spots

C E

W E

x

y

potentiostat

Cell volume : Cell volume : 30 nL30 nL

Covalent Covalent graftinggrafting in in 0.1 sec0.1 sec, , ddp2Vddp2V

spot : spot : 200200µµmm φφ, 5 nm , 5 nm thick thick

Compatibility with DNA spotters

100 spots/chip100 spots/chip

Pyrrole 20mM

Phosphate buffer

ODN-pyrrole 1µM

patent wo 02/34227, Anal Chem 2000

stainless steel electrochemical

pin

Second strategy:

liquid confining in a mobile electrochemical cellsecond generation stainless steel pin: 100 spots/chip

electrochemical cell insulated by

a teflon coating

spotsspots : : 5 to 105 to 10µµm m φφ, 5 nm , 5 nm thick thick

Underway: dedicated microfluidics

for filling and wasching

Potentiallity of more than 1000 spots/chip

electrochemical cantilevers

Second strategy:

liquid confining in a mobile electrochemical cellminiaturization and paralellization of the process: 1000 spots/chip

b

Fill-up

-E

+E

∆∆∆∆∆∆∆∆E = 2VE = 2V

Dropdeposition

Electro-deposition

Set-back

a b

100 µm

10 µm

E Descamps, P Mailley

Coll. L Nicu, LAAS Toulouse

APL 2006,

More information about the interaction ?Discrimination by temperature modulation

• control of the temperature on a DNA chipprecision + 0.02°C , slope 10°/min

Fiche et al Biophys. J. 2007

coll Theorie SPRAM DRFMC

Discrimination by temperature modulation

• hybridization of mutated DNA on the DNA chip• melting point determination…• point mutation detection.

Fiche et al Biophys. J. 2007

coll Theorie SPRAM DRFMC

Rampe M4* après hybridation à T=30°C

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

25 30 35 40 45 50 55 60 65 70

Température

Inte

ns

ité 0 mutation

1 mutation

2 mutationsA A A

T T TA A A

G

ACT

AGT

G

AAT

G

sonde

Application to the detection ofDNA repair enzymes

•Détection of formamidopyrimidine-DNA glycosylase (FpG)

Corne et al submitted NAR

coll Theorie SPRAM DRFMC

DNA chip bearing ODNs including modified bases

NHN

N

O

O O

O

O

NH2N

DNA

DNA

8-oxo-dG (5'S) 5,8-cyclo-dA

NN

N

O

O ON

NH2

H

DNA

DNA

(B)

Corne et al submitted NAR coll DRFMC LAN D Gasparutto

Fpg/DNA interactions kinetics

Red: dsDNA without damage

Blue: 8-oxodG lesion

Green: 5’(S)- CyclodA lesion

Black : poly-pyrrole control

Grey : ssDNA spot.

Application to the detection ofDNA repair enzymes

•Release of short cutted sequences

Then hybridization

Imaging of complex system ?

Sedimentation of vesicules

visualization by SPR imaging :

Vesicule size ~40µm

Near field approach :xy resolution ~5µm ; z resolution 50 to 100 nm

construction of a lipid chip for protein/lipid

interaction measurement

Injection of

Cytochrome C (+) BSA (-)

lipid vesicles A

Gold film

Hydrophobic thiol

Lipid monolayer

lipid vesicles B

mask

s s s s s s ss ss ss s s s s s s s s ss s s s

+ + + + +_____

-1

0

1

2

3

4

160 170 180 190 200 210

pcdodpcpc/pg 5:1pc/pg 2.5 :1

∆ r

efle

ctivity (

%)

time (min)

-1

0

1

2

3

4

30 0 30 2 30 4 30 6 30 8 31 0

pcd odpcpcp g60 /1 2.5pcp g60 /2 5

∆re

flec

tivty

(%)

time(min)

Suraniti et al Langmuir 2007

coll univ Sao Paulo

Blood cell analysis with SPRimaging

• Construction of the antibody chip

Collaboration P Marche MB VilliersDRDC, Inserm* L Grosjean et al. Anal. Biochem 2005

antiCD3 (LT)antiCD19(LB)

Chip bearing 25 spots

N

(CH2)5

COO N

O

O

elestrospotting

+

Pyrrole-NHS

protein protein-pyrrole

Reactive products for

protein pyrrole modification

Cell analysis with SPRimagingon an antibody chip

• Separation of B and T lymphocytes

Affinity of T cells on CD3 Ab

and B cells on CD19 Ab.

Cell analysis…cell sorting

E Suraniti, Y Roupioz submitted Collaboration P Marche MB Villiers DRDC, Inserm

Conclusion• different tools to increase the efficiency of the SPRi to be used in different fields•dd

• dealing with the problem of the analytical sensitivity of the SPR• approaches using multilayer subtrate (sharper plasmon peak)?• Labelling for SPR: gold nanoparticules

near futur • increase the lateral resolution (SPR microscopy)• hyphenated technics SPR-MS…(malcolm)

a strong link with the final biological application

BIOLOGY OPTICS

(applications) (detection)

CHEMISTRY