construction et l amplification de la Les nanoparticules d or pour la réponse des Biocapteurs Souhir Boujday, Anne-Laure Morel et Claire-Marie Pradier

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1 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

2 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

3 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

4 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 wavenumbers (cm-1) souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 4 I R

5 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

6 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

7 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

8 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

9 Gold nanoparticles synthesis 0.05 Absorbance (au) 15 nm 60nm Longueur d onde (nm) souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 9

10 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

11 XP characterization of surface functionalization Optimizing grafting conditions: olvent, temperature, time (XP analysis) c 1000 kcts a f b bound to free b/f=1 b/f>1 d 2500 kcts 1000 kcts 5000 kcts b H Binding energy (ev) Binding energy (ev) 158 souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 11

12 AFM characterization of nanoparticles grafting I Optimizing grafting conditions: Temperature, time (AFM) souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 12

13 AFM characterization of nanoparticles grafting II a b c d souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 13

14 Covalent attachment of Nanoparticles? urface Enhanced Raman cattering c b Raman ignal (au) H H H H H H H a Raman hift (cm -1 ) souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 14

15 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

16 PM-IRRA results: planar immunosensor 0.04 Planar PM-RAIR signal (au) amide band area (au) anti-rigg PrA BA rigg anti-rigg rigg Wavenumbers (cm -1 ) 1300 BA PrA Amide band area increase upon protein adsorption rigg immobilization anti-rigg/rigg = PrA BA rigg antirigg souhir.boujday@upmc.fr GdR Or-Nano, Mars 2011, Toulouse 16

17 PM-IRRA results: planar vs 3D D anti-rigg PM-RAIR signal (au) amide band area (au) H H H H H H Planar PrA BA rigg anti-rigg rigg Wavenumbers (cm -1 ) 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

18 PR Results: planar immunosensor Planar 500 anti-rigg wavenumber shift (cm -1 ) anti-rigg PrA BA rigg rigg BA PrA 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

19 PR Results planar vs 3D D 500 anti-rigg H H H wavenumber shift (cm -1 ) H H H anti-rigg Planar PrA BA rigg 40 rigg PrA BA 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

20 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

21 Thank You Réunion plénière du GdR Or-Nano 16, 17 et 18 mars 2011 Toulouse