• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content
Protein-DNA Mapping using an AFM

Protein-DNA Mapping using an AFM



This is the presentation based on our propsal of the same name. The presenters were Laura Pawlikowski, John Montoya, Ken Seal, and myself. We discuss functionalizing an AFM tip with antibodies for ...

This is the presentation based on our propsal of the same name. The presenters were Laura Pawlikowski, John Montoya, Ken Seal, and myself. We discuss functionalizing an AFM tip with antibodies for protein detection and discuss several possible uses for such a device. Conceptually, proteins bound to DNA can be flowed down a nanochannel and can be detected by an AFM tip in the channel. The detection will be based on the interaction between antibodies and their antigens (the proteins).



Total Views
Views on SlideShare
Embed Views



2 Embeds 9

http://www.slideshare.net 8
http://www.slashdocs.com 1



Upload Details

Uploaded via as Microsoft PowerPoint

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
Post Comment
Edit your comment

    Protein-DNA Mapping using an AFM Protein-DNA Mapping using an AFM Presentation Transcript

    • Using AFM for mapping protein-DNA interactions
      Anthony Salvagno
      Laura Pawlikowski
      John Montoya
      Ken Seal
    • Outline
      AFM functionalization
      Fabrication of device
      Applications of device
    • Protein-DNA Interactions
      Some Examples
      Restriction Enzymes
      Cuts DNA to protect of viral infection in bacteria
      Lots of proteins bind to control gene expression
      DNA Replication
      Proteins unwind DNA and copy DNA
      From Wikipedia: Crystal Structure
      of a protein bound to DNA
    • Gene Expression: One of the many reasons to map PDI
      DNA Transcription
      Copies DNA into mRNA
      RNA Translation
      Uses mRNA to make Proteins
      Each step mediated by protein-DNA interactions
      Mutations change everything
    • AFM and DNA mapping
      Atomic force microscopy (AFM) has been used in many applications involving DNA
      mapping exonuclease activities of DNA
      regular tip in tapping mode.
      interaction between thalidomide and DNA
      studied the topography of the substance.
    • AFM and Nanofluidics
      AFM has also been incorporated with nanofluidic applications
    • AFM functionalization
      How chips have been functionalized
      UV light used to purify chips
      Attaching antibodies to chips
      Chips allowed to sit in solution allowed to incubate, allowing the antibodies to attach
      Capacitance measurements
    • Chip Design
      AFM Chip
      Nano Channel Chip
    • Basics of Photolithography
      Remove Photoresist
      Modify Wafer Surface
      Spin-on Photoresist
      Anisotropic Etch
      Expose Photoresist
      Deposit Metal
      Develop Photoresist
      Selective Plane Etch
    • AFM Fabrication
      Step 1: Deep Anisotropic Etch
      Step 2: Deposit SiO2
      Step 1: Deep Anisotropic Etch
      Step 3: Deposit Gold Contacts
      Step 2: Shallow Anisotropic Etch
      Step 4: Deposit Indium Bond Metal
      Step 3: Silicon Plane Etch
      Flip-chip Bond Wafer 1 & 2
      Step 4: Deposit SU-8 PR
      Step 5: Deposit Gold Contact
      Silicon Release Wet Etch
    • Nano Channel Fabrication
      Step 1: Deposit PMMA PR
      Step 4: Etch Hole
      Step 2: Template PMMA PR
      Step 5: Deposit Gold Contacts
      Step 3: Define Nano Channel Cover
      Step 6: Deposit Indium Bond Contacts
    • Fabricated Device
      AFM Chip
      Nano Channel Chip
    • Device Fabrication
    • Device Applications
      Rapid Detection of Protein bound DNA
      Useful for:
      Gene Expression
      Turning genes on / off
      DNA Restriction
      DNA Repair
      DNA Mutations
    • Process
      Loading the chip
      Insert digested DNA / Protein / Buffer Mixture
      Capillary Action Feeds the mixture to the AFM tip
    • Problems
      DNA / Protein mixture must enter in a head to tail fashion.
      enters sideways
      folds back on itself
      flow out of the channel
      Researcher must know preliminary data regarding the DNA sequence
      DNA enter 3' or 5' end first
      Positive / Negative results must be followed up.
    • Conclusion
      The chip offers researchers
      rapid Protein bound DNA detection device.
      Save researchers both time an money.
      Ability to conduct entire experiment on a chip
    • Questions?
      Juven-Gershon, T., Hsu, J. Y., Theisen, J. W. & Kadonaga, J. T. The RNA polymerase II core promoter — the gateway to transcription. Curr. Opin. Cell Biol., 2008, 20, 253–259 .
      Fuda, N. J.; Ardehali, M. B.; Lis, J. T., Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature 2009, 461 (7261), 186-192.
      Hodges C, Bintu L, Lubkowska L, Kashlev M, Bustamante C. Nucleosomal Fluctuations Govern the Transcription Dynamics of RNA Polymerase II. Science, 2009, 325 (626).
      Yoo Y, Hayashi M, Christensen J, Huang LE. An essential role of the HIF-1alpha-c-Myc axis in malignant progression. Annals of the New York Academy of Sciences 2009, 1177, 198-204.
      Borrelli E, Chambon P. Control of transcription and neurological diseases. Molecular psychiatry 1999, 4 (2), 112-4.
      Schott, JJ, et al.  Congenital Heart Disease Caused by Mutations in the Transcription Factor NKX2-5. Science 1998, 281 (5373), 108.
      Hori, K.; Takahashi, T.; Okada, T., The measurement of exonuclease activities by atomic force microscopy. European Biophysics Journal with Biophysics Letters 1998, 27 (1), 63-68.
      Oliveira, S. C. B.; Chiorcea-Paquim, A. M.; Ribeiro, S. M.; Melo, A. T. P.; Vivan, M.; Oliveira-Brett, A. M., In situ electrochemical and AFM study of thalidomide-DNA Interaction. Bioelectrochemistry 2009, 76 (1-2), 201-207.
      Meister, A.; Gabi, M.; Behr, P.; Studer, P.; Voros, J.; Niedermann, P.; Bitterli, J.; Polesel-Maris, J.; Liley, M.; Heinzelmann, H.; Zambelli, T., FluidFM: Combining Atomic Force Microscopy and Nanofluidics in a Universal Liquid Delivery System for Single Cell Applications and Beyond. Nano Letters 2009, 9 (6), 2501-2507.
      Martinez, J.; Yuzvinsky, T. D.; Fennimore, A. M.; Zettl, A.; Garcia, R.; Bustamante, C., Length control and sharpening of atomic force microscope carbon nanotube tips assisted by an electron beam. Nanotechnology 2005, 16 (11), 2493-2496.
      Cuerrier, C. M.; Lebel, R.; Grandbois, M., Single cell transfection using plasmid decorated AFM probes. Biochemical and Biophysical Research Communications 2007, 355 (3), 632-636.
      Conroy, P. J.; Hearty, S.; Leonard, P.; O'Kennedy, R. J., Antibody production, design and use for biosensor-based applications. Seminars in Cell & Developmental Biology 2009, 20 (1), 10-26.
      Glawdel, T.; Ren, C. L., Electro-osmotic flow control for living cell analysis in microfluidic PDMS chips. Mechanics Research Communications2009, 36 (1), 75-81.
      Cui, Y.; Wei, Q. Q.; Park, H. K.; Lieber, C. M., Nanowirenanosensors for highly sensitive and selective detection of biological and chemical species. Science 2001, 293 (5533), 1289-1292.
      Hahm, J.; Lieber, C. M., Direct ultrasensitive electrical detection of DNA and DNA sequence variations using nanowirenanosensors. Nano Letters2004, 4 (1), 51-54.
      MacBeath, G.; Schreiber, S. L., Printing proteins as microarrays for high-throughput function determination. Science 2000, 289 (5485), 1760-1763.
      Schadt, E. E., Molecular networks as sensors and drivers of common human diseases. Nature 2009, 461 (7261), 218-223.
      Star, A.; Gabriel, J. C. P.; Bradley, K.; Gruner, G., Electronic detection of specific protein binding using nanotube FET devices. Nano Letters2003, 3 (4), 459-463.
      Whang, D.; Jin, S.; Wu, Y.; Lieber, C. M., Large-scale hierarchical organization of nanowire arrays for integrated nanosystems. Nano Letters2003, 3 (9), 1255-1259.
      Xia, D. Y.; Gamble, T. C.; Mendoza, E. A.; Koch, S. J.; He, X.; Lopez, G. P.; Brueck, S. R. J., DNA transport in hierarchically-structured colloidal-nanoparticle porous-wall nanochannels. Nano Letters 2008, 8 (6), 1610-1618.
      L. Jay Guo; Xing Cheng; Chia-Fu Chou, Fabrication of Size-Controllable Nanofluidic Channels by Nanoimprinting and Its Application for DNA Stretching. Nano Letters 2004, 4 (1), 69-73.