€¦ · Giuliano Bellapadrona,1 Alexander B. Tesler,1 Dan Grünstein,2,3 Laila Hossain,2 Kikkeri...
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LSPR TRANSDUCERS FOR SENSING PROTEIN-CARBOHYDRATE INTERACTIONS Giuliano Bellapadrona, 1 Alexander B. Tesler, 1 Dan Grünstein, 2,3 Laila Hossain, 2 Kikkeri Raghavendra, 2 Alexander Vaskevich, 1 Peter H. Seeberger, 2,3 Israel Rubinstein 1 1 Dept. of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel 2 Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, 14476 Potsdam, Germany 3 Freie Universität Berlin, Institute for Chemistry and Biology, 14195 Berlin, Germany Enhancement of the signal and detection limit with Mannose-nanoparticles Signal amplification Man-NPs Introduction Ultrathin gold island films prepared by evaporation on glass slides and annealing, combined with synthetically-modified carbohydrates, are used to develop localized surface plasmon resonance (LSPR) transducers for monitoring and imaging of protein-carbohydrate interactions. Specific recognition of mannose by Concanavalin A (Con A) is presented as a model for sensing using stationary and dynamic configurations. Conclusion Simple preparation of the tranducers. Highly specific response. Determination of kinetic parameters. Low detection limit. linker Materials Concanavalin A ( ) Gold island film ( ) Mannose-linker ( ) Galactose-linker ( ) PEG-silane ( ) Wavelength (nm) Preparation of LSPR transducers Glass slides Gold evaporation Annealing* Gold island film * 10 hours at 570 C. † Nominal thickness of the initially evaporated film. 2.5 nm † 5 nm † 15 nm † 400 600 800 0.05 0.10 0.15 400 600 800 0.05 0.10 0.15 0.20 400 600 800 0.3 0.4 0.5 Wavelength (nm) 200 nm Extinction (abs.u.) Sensing of Con A – carbohydrate interactions Blocking the glass PEG-Silane Receptor SAM* Carbohydrate Recognition Con A Sensing scheme *SAM = self assembled monolayer 400 500 600 700 800 0.05 0.10 0.15 0.20 PEG-silane Galactose SAM 1 M Con A Extinction (abs. u.) Wavelength (nm) 400 500 600 700 800 0.05 0.10 0.15 0.20 PEG-silane Mannose SAM 1 M Con A Extinction (abs. u.) Wavelength (nm) LSPR HRSEM Mannose SAM Galactose SAM FTIR Optimization Con A binding kinetics 0 300 600 900 1200 1500 0.00 0.01 0.02 Buffer Buffer Extinction 545 nm (abs. u.) Time (sec) Mannose SAM Galactose SAM 0.5 M Con A k on = 2.0 10 4 M -1 s -1 , k off = 2.6 10 -3 s -1 ext t ext [1 exp ( t/ )] on off 1/ k Con A k 0.00 0.02 0.04 0.06 0.08 0.10 0.12 (A) (B) (C) (D) 5 nM Con A 50 nM Con A 500 nM Con A No Con A 1.5 M BSA 1.5 M BSA 1.5 M BSA Extinction (abs. u.) BSA + Con A Man-NPs 1.5 M BSA + Con A + Con A
Transcript of €¦ · Giuliano Bellapadrona,1 Alexander B. Tesler,1 Dan Grünstein,2,3 Laila Hossain,2 Kikkeri...
LSPR TRANSDUCERS FOR SENSING
PROTEIN-CARBOHYDRATE INTERACTIONS
Giuliano Bellapadrona,1 Alexander B. Tesler,1 Dan Grünstein,2,3 Laila Hossain,2 Kikkeri Raghavendra,2
Alexander Vaskevich,1 Peter H. Seeberger,2,3 Israel Rubinstein1
1 Dept. of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel2 Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, 14476 Potsdam, Germany
3 Freie Universität Berlin, Institute for Chemistry and Biology, 14195 Berlin, Germany
Enhancement of the signal and detection limit with Mannose-nanoparticles
Signal amplification
Man-NPs
IntroductionUltrathin gold island films prepared by evaporation on glass slides and annealing, combined with synthetically-modified carbohydrates, areused to develop localized surface plasmon resonance (LSPR) transducers for monitoring and imaging of protein-carbohydrate interactions.Specific recognition of mannose by Concanavalin A (Con A) is presented as a model for sensing using stationary and dynamic configurations.
Conclusion
Simple preparation of thetranducers.
Highly specific response.
Determination of kineticparameters.
Low detection limit.
linker
Materials
Concanavalin A( )
Gold island film( )
Mannose-linker ( )
Galactose-linker ( )
PEG-silane ( )
Wavelength (nm)
Preparation of LSPR transducers
Glass slides
Gold evaporation
Annealing* Gold island film
* 10 hours at 570 C. † Nominal thickness of the initially evaporated film.
2.5 nm†
5 nm†
15 nm†
400 600 800
0.05
0.10
0.15
400 600 800
0.05
0.10
0.15
0.20
400 600 800
0.3
0.4
0.5
Wavelength (nm)
200 nm
Extinc
tion
(abs.
u.)
Sensing of Con A – carbohydrate interactions
Blocking the glass
PEG-Silane
ReceptorSAM*
Carbohydrate
Recognition Con A
Sensing scheme
*SAM = self assembled monolayer
400 500 600 700 800
0.05
0.10
0.15
0.20
PEG-silane
Galactose SAM
1 M Con A
Extinctio
n (
abs. u
.)
Wavelength (nm)
400 500 600 700 800
0.05
0.10
0.15
0.20
PEG-silane
Mannose SAM
1 M Con A
Extinction (
abs. u.)
Wavelength (nm)
LSPR HRSEM
MannoseSAM
Galactose SAM
FTIR
Optimization Con A binding kinetics
0 300 600 900 1200 15000.00
0.01
0.02
Bu
ffe
r
Buffer
Extinction
54
5 n
m (
abs. u.)
Time (sec)
Mannose SAM
Galactose SAM
0.5 M
Con A
kon = 2.0 104 M-1 s-1 , koff = 2.6 10-3 s-1
ext t ext [1 exp ( t / )]
on off1/ k Con A k
0.00
0.02
0.04
0.06
0.08
0.10
0.12
(A)
(B)
(C)
(D)
5 nM Con A 50 nM Con A 500 nM Con ANo Con A
1.5 M BSA 1.5 M BSA 1.5 M BSA
Extin
ctio
n (
ab
s.
u.)
BSA + Con A
Man-NPs
1.5 M BSA
+ Con A
+ Con A
