1 / 45

Development and Optimization of Quantum Dot-Neuron Interfaces

Development and Optimization of Quantum Dot-Neuron Interfaces. Jessica Winter. Prosthetics. Retinal Implant. Boston Retinal Implant Project http://www.bostonretinalimplant.org/. Therapeutics. Deep Brain Stimulator http://www.medtronic.com/. Microelectrode Arrays.

malini
Download Presentation

Development and Optimization of Quantum Dot-Neuron Interfaces

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Development and Optimization of Quantum Dot-Neuron Interfaces Jessica Winter

  2. Prosthetics Retinal Implant. Boston Retinal Implant Project http://www.bostonretinalimplant.org/ Therapeutics Deep Brain Stimulator http://www.medtronic.com/ Microelectrode Arrays. P. Fromherz, et.al. PNAS, 98, p. 10457. Biosensors Interfacing Neurons and Electronics

  3. 10 nm Voltage-Gated Ion Channel Structure and Signal Propagation. Adapted from Figure 21.13, H. Lodish, Molecular Cell Biology, W. H. Freeman & Co, 2000. Neuronal Signal Propagation Transmembrane Channel Proteins in Neurons. Adapted from Figure 21.2, H. Lodish, Molecular Cell Biology. W. H. Freeman & Co, 2000.

  4. nm Cell Diameter Cell Nucleus Ion Channel Retinal/Cochlear Implant Electrode 20 μm E-Beam Lithography Nanowire Quantum Dot Cell Receptors and Electronics Ion Channel. Adapted from www.mhhe.com/biosciesp/2001_gbla/folder_structure/ce/m3/s1 Nanocrystals (Yellow) Bound to Nerve Cell Receptors. JO Winter, TY Liu, BA Korgel, CE Schmidt. Adv Mats 13, 1673, 2001. Nerve Cell on FET Microelectrode Array. P Fromherz. Chem Phys Chem. 2002, 3, 276

  5. Our Goal Exploit nanocomponents to create functional interfaces that directly interact with nerve cell receptors

  6. 5 nm Peptide HOOCH2CS SCH2COOH Core HOOCH2CS Peptide Shell Peptide SCH2COOH SCH2COOH Quantum Dots TEM of Nanoparticle. Lattice planes demonstrate crystallinity. CdS Passivated With Ligands. Ligand Passivationelectrically insulates the crystal and provides water solubility. Core-Shell Nanocrystal. The addition of a shell layer can further passivate the particle.

  7. HEAT Quantum Dot-Cell Communication Dipole Moment Y Wang, N Herron. J Phys Chem 95:525, 1991. Light 380 nm Electron Transfer Y Nosaka, et.al. Langmiur 11(4): 1170, 1995. e- + - Heat Transfer SR Sershen, et. al. JBMR 51: 293, 2000. Cell Quantum Dot

  8. Quantum Dot Optical Properties • Size Tunable Fluorescence • Photostability • Narrow Bandwidth Bioconjugated Quantum Dots. WCW Chan, S Nie. Science 281, 2016, 1998. In Vivo Biolableing ME Akerman et. al. PNAS. 99, 12617, 2002. Multi-Labeling using Quantum Dots. Bruchez, Jr., AP Alivisatos, et. al. Science 281, 2013, 1998.

  9. Quantum Dot Dipole Moment CdSe dipole moment ~ 32 Debye Alivisatos, et. al. J Phys Chem 97:730, 1992. Nanoparticle Dipole Interaction with Ion Channels. The dipole moment produced by particle optical excitation may be strong enough to elicit a membrane voltage potential change.

  10. Debye Screening Debye Screening of the Dipole-Induced Electric Field. The electric field is screened by ions in solution, reducing the effective dipole moment.

  11. System Requirements • Efficient Electrical Interactions • Cell Attachments • Measurement of Cell Electrical Response Particle Size Ion Channel ~ 10 nm

  12. Aqueous Biologically Compatible Easily Passivated Undesirable Surface Defects Organic Few Surface Defects High Quantum Yield Requires Ligand Exchange for Biocompatibility Step 1: Chemicals Ligand Step 2: Chemicals Vacuum Injection Port N2 Quantum Dot Synthesis

  13. CdCl2 MAA Na2S NaOH pH  ~ 2 Precipitate forms pH  ~ 8-9 Qdots Form pH  7 Precipitate Dissolves Qdot Synthesis

  14. Ligand:Cd Ratio pH Cd:S Ratio Size Ligand Length Size Nanocrystal Size Dependence JO Winter, N Gomez, S Gatzert, CE Schmidt, BA Korgel. Submitted to J. of Colloids and Surfaces A, 2004

  15. Cd:S Ratio pH Ligand:Cd Ratio PL Quantum Yield PL Quantum Yield. Quantum Yield is maximum for intermediate sized particles ~ 2 nm. JO Winter, N Gomez, S Gatzert, CE Schmidt, BA Korgel. Submitted to J. of Colloids and Surfaces A, 2004

  16. Rapid Crystal Growth Reduced Passivation PL Quantum Yield Larger Smaller Particle Size Growth Mechanism Nanoparticle Quantum Yield As a Function of Particle Size. JO Winter, N Gomez, S Gatzert, CE Schmidt, BA Korgel. Submitted to J. of Colloids and Surfaces A, 2004

  17. Separation Distance System Requirements • Efficient Electrical Interactions • Cell Attachments • Measurement of Cell Electrical Response Ion Channel ~ 10 nm

  18. QUANTUM DOT 30 nm SECONDARY ANTIBODY ANTI-INTEGRIN ANTIBODY LIPID BILAYER Antibody Attachment to Cells. (To scale) Antibody Conjugation Adapted from Figure 106, Hermanson, Bioconjugate Techniques, Academic Press, 1996. JO Winter, TY Liu, BA Korgel, CE Schmidt. Adv Mat 13(22): 1673, 2001.

  19. Antibody-Directed Binding Absorbance data from antibody-qdot complexes. Complexes (dashed) demonstrate both antibody (squares) and qdot peaks (solid). Quantum Dot-Neuron Binding. (A) Phase contrast and (B) fluorescence images of antibody-conjugated quantum dots. (C) Control using fluorescent dye. (D,E) Controls with (D) no primary antibody or (E) unconjugated particles. JO Winter, TY Liu, BA Korgel, CE Schmidt. Adv Mat 13(22): 1673, 2001.

  20. Cysteine - C QUANTUM DOT 3 nm C G G G G D R S RGDS PEPTIDE INTEGRIN RECEPTOR LIPID BILAYER Peptide Attachment to Cells. Peptide Conjugation GGG- Glycine-Glycine-Glycine Hydrogen R Group, Reduces Steric Hindrance RGDS- Arginine-Glycine-Aspartic Acid- Serine Binds Cell Surface Receptors: Integrins JO Winter, TY Liu, BA Korgel, CE Schmidt. Adv Mat 13(22): 1673, 2001.

  21. Spectra of Peptide Qdots Absorbance Spectra of MAA-Quantum Dots (solid), RGD/MAA-Quantum Dots (dashed), and RGD-Quantum Dots (). FTIR Spectra of Free Peptide (Black) and Peptide Conjugated Nanocrystals (Red). Fluorescence Anisotropy of MAA-Quantum Dots (Bare) and RGD/MAA-Quantum Dots (Qdots + Peptide). JO Winter, TY Liu, BA Korgel, CE Schmidt. Adv Mat 13(22): 1673, 2001.

  22. Peptide-Directed Binding Phase Contrast (left) and Fluorescence (right) images of RGD/MAA-Quantum Dot Binding to Neurons. Fluorescence images of YIGSR/MAA-Quantum Dot Binding to Neurons. JO Winter, TY Liu, BA Korgel, CE Schmidt. Adv Mat 13(22): 1673, 2001.

  23. Non-Specific Binding Difficult to Bind Ion Channels - Highly Glycosylated - Binding Affects Channels - Bind Multiple Channel Types - Cooperative Interactions Labeling Ion Channels

  24. pH 8 pH 6 pH 7 pH 11 pH 9 pH 10 2:1 6:1 4:1 1.5:1 1:1 Non-Specific Binding Non-Specific Binding vs. Nanocrystal Reaction pH. Increased reaction pHs produce larger particles. Non-Specific Binding vs. Nanocrystal Cd:Ligand Ratio. 10 mM Ligand Concentrations (control = 55 mM) produced some non-specific binding. Non-Specific Binding vs. Nanocrystal Cd:S Ratio. Decreased Cd:S ratios produce larger particles.

  25. Cell Surface Binding Endocytosis Cell Surface Binding Peptides Antibodies Non-Specific Interactions Adapted from Figure 17-46, Lodish, Molecular Biology of the Cell, WH Freeman Company, 2000.

  26. m m 10 Endocytosis Fluorescence Images of (A) RGD-CdS Quantum Dots, (B) RGD-CdTe Quantum Dots, and (C) Non-Specifically Bound CdTe Quantum Dots at 37ºC.Endocytotic vesicles are evident in the cell interior. Fluorescence images of YIGSR/MAA-Quantum Dot Binding to Neurons at 4ºC.

  27. Day 0 Day 2 Day 1 Effect of Nanoparticle Initial Cadmium Concentration on Quantum Dot Film Fluorescence. [Silane] = 10 mM, Reaction pH = 11. Control 10 mM 15 mM 20 mM Day 6 Day 4 Day 5 m 10 m m 10 m Stability of Silane-Tethered Quantum Dot Films in Cell Culture Medium. (Fluorescence Images) Silane-Quantum Dot Films

  28. Day 0 Day 1 Day 3 Day 5 Day 4 10 m m Silane-Quantum Dot Films Stability of Silane-Tethered Quantum Dot Films in Cell Culture. (Fluorescence and phase contrast images)

  29. 20 mM m 10 m Control 2 mM 5 mM Day 5 Day 2 Day 6 Day 4 Day 0 10 mM 20 mM 15 mM m 10 m 10 m m Poly-D-Lysine-Quantum Dot Films Effect of Increased Nanoparticle Concentration on Quantum Dot Film Fluorescence. [Polylysine] = 0.5 mg/ml Stability of Polylysine-Tethered Quantum Dot Films in Cell Culture Medium. (Fluorescence Images)

  30. Poly-D-Lysine-Quantum Dot Films Day 1 Day 3 Day 0 Day 5 Day 4 10 m m Stability of Polylysine-Tethered Quantum Dot Films in Cell Culture. (Fluorescence and phase contrast images)

  31. Separation Distance Microelectrode Array. P Fromherz. Chem Phys Chem. 2002, 3, 276 System Requirements • Efficient Electrical Interactions • Cell Attachments • Measurement of Cell Electrical Response Particle Size Ion Channel ~ 10 nm Whole-Cell Clamping. http://www.bio.psu.edu/People/Faculty/Assmann/Lab/techniques.html

  32. Oscilloscope Microscope Laser Micropositioner Preamplifier Computer Microelectrodes Cell Whole-cell Clamping System. Whole-cell Clamping

  33. Day 0 Day 1 Day 4 Day 3 10 m m Whole-Cell Clamping Challenges Cell Surface Binding and Endocytosis Tethered-Film Longevity (Oxidation)

  34. Ligand Ligand Ligand Biocompatible Shell Core Ligand Inorganic Shell Ligand Ligand Ligand Biological Quantum Dots

  35. Conclusions • Antibodies and peptides may be used to create interfaces with cell receptors • Non-specific binding to cell surfaces appears to be linked to decreased ligand coverage • Recognition molecule and non-specific binding methods are subject to endocytosis

  36. Conclusions • Quantum Dot-Tethered Films May Prevent Particle Endocytosis • Films are not stable, subject to Ostwald ripening, perhaps through oxidation • Film stability is lower in cell culture

  37. Alternative Materials Aligned Particles Crosslinked Ligands Asymmetrical Particles Nanowires NH NH Genetic Mutation Phage Display Resistance to Oxidation O O Novel Binding Techniques S S S Phage S Fab Fragment + + + + + + Peptide + + + + + + + + Au Multidentate Ligands G F P + + + + Conducting Shells + + + + + + Future Directions Must improve interface longevity to allow for measurement of channel-nanoparticle interaction

  38. Therapeutics Prosthetics Ion Channels and Signal Tranduction. B. Alberts. Molecular Biology of the Cell. 4th Ed. Garland Science: New York, 2002, pp. 650. Retinal Implant. Boston Retinal Implant Project http://www.bostonretinalimplant.org/ Biosensors Ion Channel Captured Nanoparticles. T. Aida. et. al. Nature, 423, 628, 2003 Cell-Receptor Devices

  39. Acknowledgements Personal Funding NSF IGERT Fellowship NSF Graduate Research Fellowship Collaborators Dr. Richard Morrisett Dr. Adam Hendricson Graduate Students Natalia Gomez Felice Shieh Project Funding Texas Higher Education Coordinating Board (ARP) NSF Undergraduates Tim Liu Sam Gatzert Sheila Chin Facilities Center for Nano and Molecular Materials (Welch Foundation) Texas Materials Institute Advisors Dr. Christine Schmidt Dr. Brian Korgel

  40. Control 20 mM 1 mM 50 mM 10 mM m 10 m 10 mM 20 mM 2 mM 15 mM 5 mM Control m 10 m Control 10 mM 20 mM 15 mM m 10 m Day 4 Day 6 Day 1 Day 5 Day 2 Day 0 m 10 m Day 1 Day 0 Day 3 Day 4 Day 5 m 10 m Silane-Quantum Dot Films Effect of Increased Silane on Quantum Dot Film Fluorescence. [CdS] = 5 mM, Reaction pH = 7 Effect of Increased Nanoparticle Concentration on Quantum Dot Film Fluorescence. [Silane] = 10 mM Effect of Nanoparticle Reaction pH on Quantum Dot Film Fluorescence. [CdS] = 5 mM, Reaction pH = 11. Stability of Silane-Tethered Quantum Dot Films in Cell Culture Medium. (Fluorescence Images) Stability of Silane-Tethered Quantum Dot Films in Cell Culture. (Fluorescence and phase contrast images)

  41. _ _ + _ + + _ _ _ _ _ + _ _ + + + _ _ _ _ _ _ _ + + _ _ + _ _ + + Reactant Concentrations Absorbance and Photoluminescence Spectra of CdS Particles at Increasing Reactant Concentration  Concentration Charge Screening of the Solvent May Reduce Particle Solubility at Increased Concentration. JO Winter, N Gomez, S Gatzert, BA Korgel, CE Schmidt. Submitted to J. of Colloids and Surfaces A, 2004

  42. Rapid Crystal Growth Reduced Passivation PL Quantum Yield Larger Smaller Particle Size Growth Mechanism Nanoparticle Quantum Yield As a Function of Particle Size. (A) Onset of Absorption vs. pH for Neutral (), Acidic (□) and Basic () ligands. (B) Overall solubility of CdS in water vs. pH. (C) Concentration of CdS equilibrium species vs. pH. JO Winter, N Gomez, S Gatzert, BA Korgel, CE Schmidt. Submitted to J. of Colloids and Surfaces A, 2004

  43. Two point probe Electrode Bond pad Cell Bond pads Cloning ring Light 380 nm mV Time Quantum dots Microelectrode Arrays

  44. 10 mm x 10 mm Silicon Chip 185 um 185 um 2 X 1 mm 20 x 20 um 2 X 2 mm Electrode Bond Pad Bond Pad Insulated by PMMA Alignment Mark Gold Bond Pad Microelectrode Array Design

  45. Microelectrode Array Results SEM of De-Insulated Electrode (White). PMMA (gray) supports cell growth on Silicon (white) as seen in SEM.

More Related