125 5 25 1 2 3 4 5 6 University of Crete Department of Chemistry Laboratory of Analytical Chemistry Iraklion, Crete, GREECE Top Down Vs Bottom Up Approach in Bio-Sensors Nanomaterials in the Design of Chemical Sensors and Biosensors: A bottom up Approach Nikos A. Chaniotakis The Controlling Parameters of Bio-Sensors Disciplines Involved in the Design of Bio-Sensors Cost Selectivity Detection Limit Nanomaterials Reproducibility Stability Sensitivity MATERIALS Polymers Nanoparticles Semi-conductors CHEMISTRY Organic Physical Inorganic Macromolecular DEVICES BIOLOGY DNA s Cells Bio-Sensors Schematic Diagram of Bio-Sensors Nanomaterials in Bio-Sensors Analyte Electrode Nanomaterials Display Nanomaterials must have unique and novel physical and/or chemical characteristics which can aid in the design of bio-sensors with improved analytical characteristics: Signal Conditioning: Potential, ti Current, Impedance, Light Signal Transduction High surface ratio Novel electro-optical properties Increased catalytic activity Enhanced electron transfer Analyte Recognizing System, Ionophore 1
Nanomaterials in Bio-Sensors Operational Principles of Biosensors The example of Oxidase High surface ratio Novel electro-optical properties Increased catalytic activity Enhanced electron transfer Immobilization matrices Stabilization matrices Optical & electrochemical Mediators Transduction platforms +8 mv H 2 O 2 FAD oxidase Nano Materials Quantum Dots e- O 2 FADH Gluconic acid Nanomaterials Quantum Dots? Nanomaterials in Bio-Sensors Stabilization in Nano Spaces Materials Immobilization and stabilization of proteins and other biological molecules -22-2 GaN Quantum Dots Functionalization with inorganic and biological molecules l (mv) Potential -18-16 -14 3 6 9 12 15 18 21 24 Time (days) M. Vamvakaki, N.A. Chaniotakis, Anal. Chim. Acta 32 (1996) 53-61 Stabilization of Proteins in Confined Spaces Effect of confinement on the folding free energy as a function of the cage size Protein and Cage Size Maximum stabilization of proteins in spherical cages with diameter of 2 to 6 times the diameter of the native protein Active Surface Ν = 1 Ν = 2 The radius of the protein in the native state (a N ) was given by 3.73N 1/3 Cage size (in units of 2a N ) is given on a log scale. H.X. Zhou, K.A. Dill Biochemistry, 21, 4 (38), 11289 Gluconic Acid ~7 nm ~2-1 nm with polyelectrolyte 2
Stabilization Pesticide Biosensor Stabilization of Oxidase into nanoporous carbon Peripheral Site Acetylcholine W279 AChE Acylation Site W84 Acetylcholine receptors O Cl C CH O P Cl O OMe OMe O O2 N Dichlorvos P OCH 3 OCH 3 Paraoxon-methyl V. Gavalas, N.A. Chaniotakis, Anal. Chim. Acta 2, 44, 67 Porous Carbon Pesticide Biosensor Nano Biosensors Mutant (E69Y, Y71D) Drosophila melanogaster AChE +35 mv 25 oc 12 free m-ache m-ache in carbon nanopores 7 dichlorvos paraoxon 6 % Inhibition 1 8 6 4 3 1 2 2 4 6 8 Lipids 5 2 4 8 1 12 14 16 18 2 -log[pesticide], M time (hr) fluorescent indicator porin enzyme Insertion of the porin OmpF in the liposome membrane to allow substrate entrance Encapsulation of AChE in liposomes Encapsulation of the ph sensitive fluorescent indicator, pyranine substrate The enzymatic reaction lowers the ph value which is correlated to substrate concentration AChE Acetylcholine + H2O S. Sotiropoulou, N.A. Chaniotakis, Biosens.Bioelectron. 25, 2, 2347 S. Sotiropoulou, N.A. Chaniotakis, Anal.Chim. Acta 25, 53, 199 choline + acetic acid B. Chaize, M. Winterhalter, D. Fournier, BioTechniques 23, 34, 1158 Pesticide Biosensor Fullerenes Calibration Curve Fullerene C6 9 Detection Limit:7.5 x 1-11 M 8 ¾ multiple redox states ¾ low solubility in aqueous solutions ¾ stable in many redox forms 7 6 I (%) % Remaining A Activity 14 3 ± 4 nm Calibration Curve Continuous Operation 5 4 +35 mv 3 oxidase Mediator(red) 2 FAD FADH Gluconic acid 1 6 7 8 9 1 11 12 -log[dichlorvos], M V. Vamvakaki, N.A. Chaniotakis, Anal. Chim. Acta submitted emediator(ox) 3
Fullerenes Fullerenes +1mV +35mV Fullerene Mediator Oxidase FAD Hydrodynamic voltammogram for the glucose biosensors constructed using carbonincubatedfor:( ),4( ),5( ) cycles in the toluene-c 6 solution Calibration curve of the glucose biosensor containing 1.7µg C 6 /mg of electrode material. Measurements were performed in 1mM phosphate buffer, ph=7.5 under argon, at +35mV vs. Ag/AgCl. e - FADH Gluconic acid Flowchart of the processes involved in a light induced fullerene mediated electrochemical biosensor. The operating potential has dropped to +1 mv. V. Gavalas, N.A. Chaniotakis, Anal. Chim. Acta 2, 49, 131 Fullerenes Carbon Nanotubes -.4 -.2 Pt Transducer Gluconic acid. Ι (µα Α).2.4.6 Light ON Light OFF e - Oxidase.8-1 1 2 3 4 5 [], mm The carbon nanotubes were grown by the CVD method on a platinum substrate, thus providing an array of MWNT, 15-2 microns long and with an internal diameter of 15nm. S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 23, 375, 13 Carbon Nanotubes Carbon Nanotube Biosensor SEM images of the Carbon Nanotubes 2. 1.5 Linear range:.5-2.5 M Sensitivity: 93.9 ±.4 µa mm -1 cm -2 (µα) Ι 1..5 Initial Carbon Nanotube Array Acid oxidation (HNO 3 /H 2 SO 4 ) Air oxidation (6 C, 5min)...5 1. 1.5 2. 2.5 [glucose] (mm) S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 23, 375, 13 S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 23, 375, 13 4
Carbon Nanofiber Biosensor Carbon Nanofiber Biosensor Table 1. Carbon nanofiber physical characteristics Nanofiber Grade LHT HTE GFE Diameter (nm) 7-15 8-15 8-15 N2 Surface Area (m 2 /g) 43 8-1 > 5 Density (g/cm 3 ) > 1.95 1.98 2.17 Heat treatment ( o C) 1 1 3 Metal Content (wt. %) <.5 <.5 <.1 Electrical Resistivity (Ohm/cm) < 1-3 < 1-3 < 1-3 SEM image of HTE Nanofibers mean diameter ~ 11 nm length ~ tenths of nanometers Carbon Nanofibers Carbon Nanotubes Carbon Nanofiber Sensor Carbon Nanofiber BioSensor I (A A) 1.x1-3 5.x1-4. -5.x1-4 -1.x1-3 GFE HTE LHT NANOTUBES GRAPHITE -.8 -.6 -.4 -.2..2.4.6.8 1. 1.2 E (V) ing Activity % Remaini 15 14 13 12 11 1 9 8 7 GFE HTE LHT NANOTUBES GRAPHITE Stability Study 2 4 6 8 1 t (hours) Reproducibility: RSD value < 1% (N = 3) GaN Quantum Dots By altering the particle size and the chemical composition of the QDs the fluorescent emission changes. Quantum Dots A quantum dot 5
Optical Properties of GaN quantum dots Conclusions-Future Directions Intensit ty (a. u.) 11 1 9 8 7 6 5 4 3 2 1 Photoluminescence spectra GaN QDs GaN QDs - KCl 1M GaN QDs - KCl 2M 4 45 5 55 6 65 7 75 Wavelength (nm) Depending on the KCl concentration Blue shift Rise of intensity Nanomaterilas have unique properties that are ideal for the development of highly stable, reproducible, and sensitive chemical sensors and biosensors The particle size and the chemical composition altered QDs fluorescent emission changes Acknowledgments Sofia Sotiropoulou Vicky Vamvakaki Maria Fouskaki Jiannis Alifragis Antonis Volosirakis Kleri Karapidaki Colaborations Microelectronics Group FORTH Prof. Ambacher and his group TUI This work is being supported by the European Commission Programs GANANO and SAFEGARD, IRAKLITOS and ARCHIMIDIS of the Greek Ministry of Education. 6