The sweet surface of a cell The outer 100 nm

Transcription

1 The Sweet Science of Glyco-Nanotechnology Freshman Seminar Gen ST 197 D Nanotechnology & Molecular Engineering Daniel M. Ratner Dept. of Bioengineering University of Washington The sweet surface of a cell The outer 100 nm ~100 nm From Biochemistry, Voet and Voet 1

2 Synthetically-Derived Tools for Glycomics Ratner, Adams, Disney and Seeberger. ChemBioChem Developing Label-Free Glycoarray Platforms Complementary Metal-xide Semiconductor (CMS) Imaging SPR Fluorescent Microarray Nanophotonic Devices 2

3 EG-Thiol Linking Chemistry EG Ensure carbohydrate ligand is sufficiently solvated Thiol Amenable to synthesis, versatile for immobilization Ratner, D. M.; Adams, E. W.; Su, J.; Keefe, B. R.; Mrksich, M.; Seeberger, P. H. Probing Protein- Carbohydrate Interactions with Microarrays of Synthetic ligosaccharides. ChemBioChem. 2004, 5, Screening the High-Mannose Array Ratner, Adams, Disney and Seeberger. ChemBioChem

4 Antimicrobial specificities Cyanovirin-N Scytovirin Adams, Ratner, Seeberger et al. Chem. Biol Significance of Glycan Surface Density Monovalent binding Multivalent binding Multivalency / Cooperativity Antigenicity Pathogen binding/inhibition 4

5 Model Array Surface: Mixed SAMs on Gold Dhayal and Ratner. Langmuir, ToF-SIMS Provides fragment ion mass spectrum of a surface Exquisitely surface sensitive (top 15 Å) High resolution depth profiling Able to image features down to ~5 μm Can distinguish immobilized sugars NESAC/BI 5

6 Everything you wanted to know about your surface chemistry, and more Total ions Na + K + C 2 H 5 + (SAMs) C 4 H 6 N 3 + (NHS) NH 4 + (GDAP) C 5 H 5 N 3 + (GDAP) 100 μm XPS / ESCA Establish reference for glycan surface modification Composition of glycoarray surface Quantify mixed SAMs NESAC/BI 6

7 ESCA Analysis of EG/Carbohydrate SAMs Label-Free ptical Glycoarray Platforms SPR Imaging Fluorescent Microarray Silicon Nanophotonics 7

8 Plasmon Resonance Where have we seen SPR before? 8

9 H H H H 2/8/10 SPR Imaging on the GWC SPRImager II Model SAMs for SPR Imaging Analysis 9

10 SPR of Mixed SAMs with Alkanethiolate EG SPR of Mixed SAMs with Alkanethiolate EG 10

11 SPR of Mixed SAMs with short EG-thiol ConA-binding Seeing is believing Feeling Microscopy Scanning Probe Microscopy 11

12 Scanning Probe Microscopy Scan lines y7 y6 y5 y4 y3 y2 y1 height (z) -> brightness mrsec.wisc.edu Atomic Force Microscopy piezoelectric positioners mrsec.wisc.edu 12

13 Can we visualize sugars assembled on the surface? EG Sugar vs. Gold Gold Scaling sugars in nanometers Sugar = ~1.9nm Linker= ~1 nm 13

14 Can we use these tools for Biology?! Bacterial Adhesion Structure of E. coli Adhesin FimH 50 nm H H 500 nm = ½ μm H H H Cell Surface H > 90% of E. coli and other enteric bacteria have Type I Pili *Courtesy Wendy Thomas (UW, Bioengineering) 14

15 Limited Range of Surface Plasmon (~200 nm) Surface Plasmon nm Surface plasmon extends ~200 nm Pili limit effectiveness of SPR for direct detection How can we extend the range of detection? SPR Imaging Fluorescent Microarray Silicon Nanophotonics 15

16 Silicon Photonics: Extending the Range of Label-Free Detection 1 micron 1 micron High surface field density World s premier material for making nanostructures Billions of dollars of infrastructure for making silicon devices *Courtesy Michael Hochberg (UW, Electrical Engineering) They call it Nanophotonics for a reason Human Hair 1 micron 100 microns *Courtesy Michael Hochberg (UW, Electrical Engineering) 16

17 The Silicon Nanophotonic Ring Resonator Whispering-gallery-mode biosensor Evanescent wave-based detector, range nm In collaboration with M. Hochberg and Genalyte, Inc. (Photo credit, M. Hochberg) RR vs. SPR: Pathogen Sensing 17

18 Ring Resonator Glycoarray (Photo credit, M. Hochberg) Silicon photonics scales readily 10,000+ devices in single chip Limited by fluidics and sample handling 18