Les nanoparticules d or pour la construction et l amplification de la réponse des Biocapteurs ouhir Boujday, Anne-Laure Morel et Claire-Marie Pradier Laboratoire de Réactivité de urface, UMR 7197, Université Pierre et Marie Curie CNR Réunion plénière du GdR Or-Nano 16, 17 et 18 mars 2011 Toulouse
What is a biosensor? Biosensors are defined as analytical devices incorporating a biological material a biologically derived material or a biomimic intimately Intimate associated association with or integrated within a physicochemical transducer or transducing microsystem. Biosensors & Biolectronics 2009 Antibodies, DNA, Enzymes, Aptamers etc. Metal or oxide surface gold, silicon, carbon etc. Recognizes with high affinity and selectivity the target of interest Allows measuring the molecular recognition phenomena Must guaranty an efficient immobilization of the biological element souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 2
Elaboration of Gold based biosensors Reducing response-time Increasing selectivity Increasing sensitivity High amount / good accessibility / preserved biological activity R R R R s s s s O OH R R # R R # R R # R R # s s s s s s s s Must guaranty an efficient immobilization of the biological element souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 3
Optimization (and transduction) tools : s s s s Vibrational spectroscopies IR, Raman PM-IRRA, ATR, DRIFT, ER X-ray Photoelectron pectroscopy I R Rayons X e - Atomic Force Microscopy CO amide I O 1655 cm -1 Increasing protein amount R 1 R 1 C HN R 2 IR beam at grazing incidence ignal surface enhanced No contribution of the isotropic environment s s s s CN + NH amide II 1550 cm -1 2300 2100 1900 1700 wavenumbers (cm-1) 1500 1300 souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 4 I R
And, in-situ sensitive transduction techniques Fourier Transform urface Plasmon Resonance FT-PR Coated film ensor surface ample media Glass slide Measures the wavenumber of maximum extinction of total internal reflection of light (at a fixed angle of incidence) Reflected beam P-polarized Incident beam souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 5
And, in-situ sensitive transduction techniques Quartz Cristal Microbalance with QCM-D dissipation Piezoelectric quartz disc Measures mass changes and viscoelastic properties in realtime and with nano-sensitivity Gold electrodes ensor surface Frequency shift of an oscillating quartz Mass change C f n auerbrey relation (1959) m= - C = 17.7 ng Hz -1 cm -2 for a 5 MHz quartz crystal. n = 1,3,5,7 is the overtone number. souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 6
Nanoparticles for Biosensors Either grafted on the transducer surface Nanostructuring surfaces Increasing grafting surfaces or linked to the biological material Increase sensitivity of optical techniques Increasing piezoelectric / gravimetric response souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 7
Why grafting nanoparticles? Ab amount Ab accessibility efficient immunosensor Increase Ab density Enhance flexibility Preserve accessibility H H H Increase sensitivity of optical techniques souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 8
Gold nanoparticles synthesis 0.05 Absorbance (au) 15 nm 60nm 400 500 600 700 800 900 1000 Longueur d onde (nm) souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 9
urface functionalization to graft Nanoparticles hexanedithiol : H 2 thiols extremities bound to surface free to bind nanoparticles H + Long chain (preserve flexibility) Can we avoid bridging? H b/f=1 b/f>1 souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 10
XP characterization of surface functionalization Optimizing grafting conditions: olvent, temperature, time (XP analysis) c 1000 kcts a f b 162.0 163.3 bound to free b/f=1 b/f>1 d 2500 kcts 1000 kcts 5000 kcts b 163.6 164.9 H 168 163 Binding energy (ev) 158 168 163 Binding energy (ev) 158 souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 11
AFM characterization of nanoparticles grafting I Optimizing grafting conditions: Temperature, time (AFM) souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 12
AFM characterization of nanoparticles grafting II a b c d souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 13
Covalent attachment of Nanoparticles? urface Enhanced Raman cattering c b 1072 1303 2852 Raman ignal (au) 2550 654 1435 2927 2927 1140 1265 H H H H H H H a 400 600 800 1000 1200 1400 Raman hift (cm -1 ) 2500 2700 2900 souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 14
Building of a NP-Biosensor H H H H H H NP immunosensor Planar immunosensor Mercaptoundecanoic acid Protein A anti-rigg BA rigg souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 15
PM-IRRA results: planar immunosensor 0.04 Planar 14 12 10 8 6 4 PM-RAIR signal (au) amide band area (au) anti-rigg PrA BA rigg anti-rigg rigg 2 1900 1700 1500 Wavenumbers (cm -1 ) 1300 BA PrA Amide band area increase upon protein adsorption rigg immobilization anti-rigg/rigg = 4.2 0 PrA BA rigg antirigg souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 16
PM-IRRA results: planar vs 3D 0.04 3D anti-rigg 14 12 10 8 6 4 PM-RAIR signal (au) amide band area (au) H H H H H H Planar PrA BA rigg anti-rigg rigg 2 1900 1700 1500 Wavenumbers (cm -1 ) 1300 2 times more PrA 4 times more rigg anti-rigg/rigg = 2.1 BA PrA 0 PrA BA rigg antirigg increasing Ab amount preserving their accessibility. Boujday et al. Coll. urf. (2010) souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 17
PR Results: planar immunosensor 120 110 Planar 500 anti-rigg 100 400 300 200 wavenumber shift (cm -1 ) 90 80 70 60 50 anti-rigg PrA BA rigg 40 30 20 10000 9500 9000 8500 rigg BA PrA 8000 7500 100 0 PrA BA rigg anti-rigg Wavenumber shift of maximum extinction upon protein adsorption rigg immobilization anti-rigg/rigg = 3.7 souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 18
PR Results planar vs 3D 120 110 3D 500 anti-rigg 100 90 80 70 60 50 H H H 400 300 200 wavenumber shift (cm -1 ) H H H anti-rigg Planar PrA BA rigg 40 rigg 100 30 20 10000 9500 9000 8500 PrA BA 8000 7500 0 PrA BA rigg anti-rigg 2 times more PrA 2 times more rigg anti-rigg/rigg = 2.7 increasing Ab amount preserving their accessibility souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 19
ummary and perspectives Despite particles size: enhancement of protein amount by a factor 2 and accessibility is preserved! souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 20
Thank You Réunion plénière du GdR Or-Nano 16, 17 et 18 mars 2011 Toulouse