University of Crete Department of Chemistry Laboratory of Analytical Chemistry Iraklion, Crete, GREECE URL: www.analytical_chemistry.uoc.gr Nanomaterials as Transducers in Biosensors.. Chaired by: Dr Renzo Tomellini (EC RTD.G4) Recommendations: Bioelectrochemistry 7 Toulouse 1 st to 4 th April 7 Nikos A. Chaniotakis Vicky Vamvakaki http://www.nanoforum.org/ 1 2 Nanotechnology Nanotechnology is the exploitation of novel properties of materials and systems that are manifested due to their scale: 1. Chemical (catalysis, electrochemistry, selective interaction, magneto-optical characteristics) 2. Biochemical (stabilization, signal mediation, functionalization, tagging) 3. Physical (quantum characteristics, magnetic, optical, physical) Nanotechnology Areas for Development 1. The design and synthesis of functional nanomaterials 2. Study of the chemical and physical properties 3. Use in detection (environment and safety) 4. Applications in healthcare (therapeutics and diagnostics) 5. Nano-bio-electronics, photonics, and magnetics 6. Energy conversion and storage 7. The synthesis-large scale manufacturing 8. Environmental-health impacts 3 4 The Nanotechnology Development Stages 1 st : Non-functional Nanoelectrodes, catalysts, mediators 2 nd : Functionalized- Direct generic detection, Functional nanoreactors, transistors 3 rd : Functional and Selective Selective, highly sensitive and autonomous monitoring systems Nano-Biosensor Development Our research in the area of Biosensors is directed towards the development of systems with: Improved analytical characteristics (detection limit and selectivity) and Unique detection characteristics (direct and simple transduction) 5 6 1
Nanomaterials in Bio-Sensors Nano Materials in Bio-Sensing Technology Nanomaterials must have unique and novel physical and/or chemical characteristics which can aid in the design of bio-sensors with improved analytical characteristics: Carbon Nanofibers ¾ High surface ratio Fullerenes ¾ Novel electro-optical p properties p p Nanocavities (C, Au, Ag, Si) ¾ Increased catalytic activity Nanoparticles (Pt, Au, Ag) ¾ Enhanced electron transfer Quantum Dots (semiconductors) Immobilization and Stabilization matrices, Mediators, Transduction platforms Carbon Nanotubes 7 Nano Materials in Stabilization, Transduction and Selective Interaction 8 Quantum Dots Important parameters: 1 Band gap energy of semiconductor 2 Size and shape of QD 3 Capping 4 Surface functional groups Fullerenes, CNTs, CNFs, Liposomes, Nanocavities 9 Stabilization in Confined Spaces 1 Protein Stabilization in Nanocages Effect of confinement on the folding free energy as a function of the cage size H.X. Zhou, K.A. Dill Biochemistry, 1, (38), 11289 Ν = 1 Ν = The radius of the protein in the native state (an) was given by 3.73N1/3 Cage size (in units of 2aN) is given on a log scale. Biosens. Bioelectron. 7, 22, 265 11 12 2
Protein Stabilization in Nanocages Folding free energy of proteins in 1 nm cage diameter HRP (N=36) - Toxicity Biosensor Peripheral Site Acetylcholine W279 G G/k B T - -6 GOx (N=1166) AChE Acetylcholine receptors Acylation Site W84-5 1 15 25 Ν (residues) Biosens. Bioelectron. 7, 22, 265 Cl C Cl O CH O P Dichlorvos OMe OMe O O 2 N O P OCH 3 OCH 3 Paraoxon-methyl 13 14 AChE Stabilization into Nanoporous Carbon Leaching 1 m-ache in porous carbon 1 m-ache free 1 Continuous Operation free m-ache m-ache in carbon powder AChE Stabilization into Nanoporous Carbon 7 6 Mutant (E69Y, Y71D) Drosophila melanogaster AChE dichlorvos paraoxon % Remaining Activity 6 % Remaining Activity 1 6 5 3.5 hr 2 hr 6 hr wash time 19 hr 48 hr 6 time (hr) Leaching rate Mutant Acetylcholinesterase, +35 mv, 25 o C m-ache free: 1.8%/hr m-ache in porous carbon:.7%/hr S. Sotiropoulou, V. Vamvakaki, N.A. Chaniotakis, Biosens.Bioelectron. 5,, 1674 1 8 1 12 14 16 18 -log[pesticide], M Biosens.Bioelectron. 5,, 2347 Anal.Chim. Acta 5, 53, 199 15 16 Toxicity Liposome Nano-Bioreactor Toxicity Liposome Nano-Bioreactor 3 nm porin active enzyme fluorescent indicator pesticide substrate inhibited enzyme 9 7 6 5 3 1 1 6 7 8 9 1 11 12 6 7 8 9 1 11 12 ~ 1 1 M ~1 9 M -log[dichlorvos], M -log[paraoxon], M Biosens. Bioelectron. 5, 21, 384 Biosens. Bioelectron. 7, 22 265 7 6 5 3 Fig. 6. Image of the colorimetric pesticide screening array in the presence of different dichlorvos concentrations after 15 min reaction with 1 mm substrate. Incubation with pesticide was performed for 15 min. Biosens. Bioelectron. 7, 22 265 18 3
Carbon Nanotubes Nanofibers vs. Nanotubes Glucose Gluconic acid Pt Transducer e - Enzyme Glucose Oxidase The carbon nanotubes were grown by the CVD method on a platinum substrate, thus providing an array of MWNT, 15- microns long and with an internal diameter of 15nm. S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 3, 375, 13 19 Anal. Chem.; 6; 78(15) pp 5538 Anal. Bioanal. Chem. 3, 375, 13 aining Activity % Rema Carbon Nanofiber BioSensor Stability Study 15 1 13 1 11 1 9 7 GFE HTE LHT NANOTUBES GRAPHITE 6 1 t (hours) Reproducibility: RSD value < 1% (N = 3) Anal. Chem.; 6; 78(15) pp 5538 21 +35mV +1mV e- Fullerene Mediators multiple redox states low solubility in aqueous solutions stable in many redox forms Enzyme Glucose Oxidase FAD FADH Glucose Gluconic acid From: N. Chaniotakis. In Nanomaterials for Biosensors Nanotechnologies for the Life Sciences Kumar, Challa S. S. R. (Hrsg.) 6 22 Conclusions Nanomaterials are very promising as a) mediators b) transducers and c) stabilizers in biosensors The physical and chemical properties of the material must be well known and controlled Funds 1. SAFWGARD: Sensor Arrays for Environmental, Generic and Routine Detection of Pesticides 2. GANANO: New Generation of GaN-based Sensor arrays for nano- and pico-fluidic systems for fast and reliable biomedical testing 3. SANTS: Synthesis and Application of Nanostructured Tethered Silicates 4. NANOMYC: Multiparametric detection of bio-molecule conjugated nanoparticles for the diagnostic investigation of mycobacterial infections of humans and animals 23 24 4
Thanks to: Dr. V. Vamvakaki Dr. D. Fournier Dr. S. Sotitopoulou Dr. J. Alifragis Dr. K. Kostantinidis Ms M. Fouskaki Ms. K. Tsagaraki 25 26 5