Fluorogenic Polydiacetylene Nanosomes: Application to Label-Free Nanobio Sensors Dong June Ahn ahn@korea.ac.kr, +82-2-3290-3301 Department of Chemical & Biological Engineering College of Engineering, Korea University Seoul, Korea April 17-19, 2008 D. J. Ahn, Korea University Needs for Sensors New & Mutated Infectious Diseases Diagnoses & Drug Discovery SARS Anthrax Mutated Astronomical cost Cholera, spent each year etc. worldwide ! Bio Chips & Chemical Chips Safety for Processed Food Need for Disease Monitoring D. J. Ahn, Korea University 1
Outline � Polydiacetylene Supramolecules � Detection Examples � Nanosome-Based Chips � Nanosome Strips and Fibers � Summary D. J. Ahn, Korea University Current Concept for Sensing Systems and Chips • Sensor Systems • Diagnostic Array Chips Target Substance Recognizing Materials Human Judgment A A A B C A A C B C A B B A C Labels: Cy5, Cy3, Interface I Interface II nanoparticles, radio tracers Target / Sensor Matrix Sensor Matrix / Human A T Affinity Signal Transducer G C Transistors / Optics Shape T A T Size Electrochemistry C A Mechanics / Nanowires Pre-treatments DNA Chip Protein Chip For Labeling Analytes D. J. Ahn, Korea University 2
Why Conjugated Systems? • Conventional Chemosensor T target molecule S-T S Sensitivity depends on the equilibrium constant. K eq = [S-T] / [S] [T] π -Conjugated Polymer Sensor • target molecule Signal amplification can be achieved. D. J. Ahn, Korea University π -Conjugated Polymers • π -Conjugated Polymers O O R O O R O O R O O O O R R NH HN S S R n n Polythiophene Polypyrrole Poly(phenyleneethylene) R R R N N R Combination (PPE + Bipyridil) R R Polydiacetylene Electrical and/or optical properties • Easily dispersed into aqueous media ! D. J. Ahn, Korea University 3
Polydiacetylene Supramolecules • Synthetic Diacetylene Supramolecules • Natural Cell Membranes O Hydrophobic tail Ligand inner aqueous compartment diacetylene unit Hydrophilic head molecular recognition self-assembly Langmuir-Blodgett dispersion by lipid bilayers Langmuir-Schaefer sonication hydrophilic hydrophobic solid-supported films nanosome (vesicle) solution head groups tails X X X X X X O O O O O O oligosaccharide hydrophobic glycolipid α -helix O O O O O O X X X X X X AFM SEM phospholipid integral protein cholesterol Cytosol molecular spacing : 4.8 ± 0.2 Å size : 50 nm D. J. Ahn, Korea University Optical Characteristics Red Blue Colorless h ν Stimuli X X X O X O O O O X O X O X O X O X X O X O X O X O X O O O O X X X O X Real-Time Observation D. J. Ahn, Korea University 4
Polydiacetylene-Based Sensor Systems target color change conjugated p-orbital twisted p-orbital blue red (Increased response) Blue phase Red phase (Non-fluorescent) (Fluorescent) D. J. Ahn, Korea University Detection Examples D. J. Ahn, Korea University 5
Detection of Influenza Virus A Charych et al., Science , 261, 585 (1993); J. Am. Chem. Soc., 117, 829 (1995). increase of virus concentration D. J. Ahn, Korea University Detection Examples • Influenza Virus A: Charych (1993, 1995) • Antibody: Jelinek (2001) OH HO OH sialic acid O AcHN COOH HO O NH O R 3 O O peptide O R 1 H N O OH O N O NH H O OH N R 2 5 5 H O 5 6 6 6 • Small Molecule (Cyclodextrin): • DNA: Wang & Ma (2005) Ahn & Kim (2003, 2004) pore match O NH O NH O NH 5 5 5 6 6 6 D. J. Ahn, Korea University 6
“Conceptual Evolution” O OH O OH O OH 5 5 5 6 6 6 Nanosome Solutions Films on Substrates Method: Langmuir-Schaefer integration Method: Sonication Use: Film-type Sensor Use: Solution-type Sensor Low Signal Medium-High Signal One Binding Event per Film One Binding Event per Solution NH 2 New H 2 N NH 2 H 2 N Surface Immobilized Nanosomes Embedded NH 2 Concept H 2 N NH 2 H 2 N Nanosomes N Method: Surface Chemistry H Advanced Materials, 15, 1118 (2003). N H Method: Polymer Blending Patterned Nanosomes “Nanosome Strips” Method: Microcontact Printing “Nanosome Fibers” Use: Sensor Method: Molding & Spinning One Binding Event per Patterned Nanosome Surface 200㎛ Use: Chemical & Bio Sensor Blue “Nanosome Chips” Blue Method: Inkjet Spotting Blue Use: Chemical & Biological Chip Purple Simultaneous Screening & Detection Pink Self-Emitting Fluorescence Red “Label-Free System” Advanced Functional Materials, 16, 2103 (2006). J. Am. Chem. Soc., 127, 17580 (2005). Advanced Materials, 19, 521 (2007). D. J. Ahn, Korea University Nanosome-Based Chips D. J. Ahn, Korea University 7
Nanosome Array Chips Inkjet (non-contact), single tip < Ink-jet Microarrayer > < Microarray of Nanosomes > s s a l g S P A 1 spot (4 nL), width: 200 ㎛ Conventional sol ’ n analysis: 0.1 mL • Pre-spot Nanosome Library 25,000-fold saving • Solutions in 96 or 384 well plates • Tiny amount of analytes Simultaneous multiple screening • D. J. Ahn, Korea University Microarray-Based Fluorescence Profiles α -CD (30mM ) γ -CD (30mM) γ -CD → Heating Linear Carbohydrate : Maltoheptaose(30mM) Maltoheptaose → Heating Polyacrylic acid (30mM) Ahn et. al., J. Am. Chem. Soc., 127, 17580 (2005). D. J. Ahn, Korea University 8
A Label-Free Detection of Protein-Protein Interaction initial after exposure to anti-rabbit after heated secondary antibodies when primary after heated after exposure to anti-mouse after heated antibody is absent secondary antibodies Advanced Functional Materials (2007); Macromolecular Research (2006). D. J. Ahn, Korea University A Label-Free Detection of Bacteria D. J. Ahn, Korea University 9
Detection of E. Coli PCDA-Aniline + PCDA-EDEA-s-NHS PCDA + PCDA-EDEA-s-NHS when primary antibody is absent Submitted to Angew. Chem. Int. Ed. (2007). D. J. Ahn, Korea University DNA Detection Sensitivity Spotting of vesicle-probe DNA complex 3` - GAT GGA TGC CCA TTG - 5`-NH 2 Polymerization 5` - CTA CCT ACG GGT AAC - 3` E. coli lethal factor sequence 15 mer. 200 ㎛ target DNA 250 200 Intensity (a.u.) 150 100 nM 10 nM 1 nM 100 pM 10 pM 100 50 0 100 nM 10 nM 1 nM 100 pM 10 pM 1 pM 100 fM 10 fM 1 fM -- Target DNA conc. 1 pM 100 fM 10 fM 1 fM D. J. Ahn, Korea University 10
DNA Detection Selectivity: Perfect Match vs. Single-Base Mismatch Vesicle Vesicle & probe DNA Pre spot Solution NH 2 – 5` - GTT ACC CGT AGG TAG - 3` 3` - CAA TGG GCA TCC ATC - 5` TAG 200 ㎛ A Single strand Polymerization G T Immobilization [ ] Si O Si O Si O Si O Si O Si O O O O O O O Si Si n Si Si Si Si Double strand target DNA Mismatched DNA ] Si O Si O Si O Si O Si O Si O O O O O O O Si Si Si Si Si Si n TAG G A T Filter 3. (green) D. J. Ahn, Korea University Nanosome Strips and Fibers D. J. Ahn, Korea University 11
Nanosome-Embedded Strip Films PCDA liposomes in PVA Drying Thermal Response Confocal Fluorescence Microscopic Image D. J. Ahn, Korea University “Litmus-Type” Strip Sensors for Chemicals O OH O OH O OH O OH 5 5 5 5 7 7 7 7 α -CD β -CD γ -CD control strip solution Chemistry Letters, 35, 560 (2006). D. J. Ahn, Korea University 12
Image Display Using Nanosome-Embedded Films Advanced Functional Materials, 16, 2103 (2006). after photopolymerization thru a photomask after heating after photopolymerization of the remaining area D. J. Ahn, Korea University Electrospun Polydiacetylene Fibers R 2 R 1 R 1 R 1 R 2 R 2 R 2 R 2 R 1 R 1 Randomly-distributed DA electrospinning R 2 R 2 R 2 R 2 R 1 R 1 R 1 R 1 Self-assembled DA hv R 2 R 2 R R 2 2 R R 1 1 R R 1 1 PDA-embedded fiber Advanced Materials, 19, 521 (2007). D. J. Ahn, Korea University 13
Visual Detection of Food Transmutation beef, pork hairtail, croaker, skate, etc chicken pH=11.0 pH=12.3 pH=8.8 Gas Analytes Liquid Analytes D. J. Ahn, Korea University Visual Alarm System (Example Case: Formaldehyde) HCHO detection D. J. Ahn, Korea University 14
Summary of Sensor Research Blue Blue Red Blue Blue 100㎛ + Target + Target + Target Purple - Target Pink Red 100㎛ Self-Emitting Fluorescence Color-Changing No Fluorescence Fluorescence “Label-Free Chip System” “Label-Free Strip System” Label-Free Chips and Strip Sensors Reusable or Continuous-Monitoring System D. J. Ahn, Korea University Acknowledgments My Dear Students Home Coming Day Dr. D. H. Charych (Lawrence Berkeley Nat’l Lab. / Chiron Corp. / Five Prime Corp.) Prof. Jong-Man Kim (Hanyang University) Research Funds from Korean Government (KISTEP, KRF, KOSEF, MOH) and Private Sectors 15
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