AFM Spectroscopy of Biomolecules ME382 : Micro/Nanoscale Science and Engineering Prof. Horacio D. Espinosa Winter 2004-05

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AFM Spectroscopy of BiomoleculesME382
Micro/Nanoscale Science and EngineeringProf.
Horacio D. EspinosaWinter 2004-05

• Presented by
• Ravi Agrawal, Kevin Lee, Deepak Ponnavolu

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Outline

• Introduction and Working Principle
• AFM in liquids
• Substrate Functionalization
• Tip Functionalization and Tip parameters
• Developments and Applications
• Typical force curve
• Conclusion

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Tip Sample Interaction
Schematic diagram of the vertical tip movement
during the approach and retract parts of a force
spectroscopy experiment
Tip Far Away (10-100 microns) No interactions
Tip Approaching (few microns) Electrostatic Forces
Tip Close to Surface Van der Waal
Forces Capillary Forces
Contact Tip Indenting the sample Stiffness Viscoel
astic Response
Lifting Off Surface (few atomic distances to
nanometers)
Tip farther away (nanometers to hundreds of
nanometers) Stretched molecules between tip and
surface Protein unfolding, pulling out of
membranes
Tip Far Away (1-5 microns) Connection between the
tip and substrate is broken. No more interactions
Technical Report Nanowizard A practical guide
to AFM force spectroscopy and data analysis
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Working Principle

• Linker molecules are used to functionalize
  the tip and the substrate
• Ligand and receptor molecules are attached
  to the linker molecules
• The functionalized substrate holding the
  receptor molecule is approached with AFM tip
• Ligand recognizes the receptor and fits in
• AFM tip can be retracted to study the
  response of the ligand-receptor molecule

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AFM in Liquids Biological Applications

• Why in Liquid ?
• To prevent contamination of sample from
  hydrocarbons present in air
• Most of the biomolecules are active in aqueous
  environment
• Most of the cells (except few epithelial ones)
  die in dry conditions
• AFM in liquids
• Higher amplitude of oscillation to get good
  signal to noise ratio
• Quality factor (wom/b) goes down drastically
  because of high hydrodynamic damping
• Typical thickness of cantilevers in tapping mode
  10 microns. By increasing this thickness,
  Q-factor as well as stiffness can be increased to
  an optimum value
• Included angle of the tip should be lower in
  order to cut down the van der Waals forces

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Substrate Functionalization

• Why do we need to functionalize the substrate ?
• To hold the molecules onto the substrate
• To align the molecules in a particular way to
  maintain selectivity

DNA motion during scanning (AC). AFM images in
TE buffer over a 300 300 nm area. The images in
AC were taken with an interval of 4.5 min
http//www.pubmedcentral.gov/articlerender.fcgi?t
oolpmcentrezartid19541
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Substrate Functionalization

• Binding of biological molecules to a solid
  substrate
•     poly-L-lysine or poly-L-arginine
•     silanizing a solid surface with
  3-aminopropyltriethoxysilane (APTES)
•     Cross-linking group via the amino end of
  APTES on a glass surface (ANB-NOS).
•     ultraflat Au(111) surface is used as a
  substrate for N-hydroxysuccinimide terminated
  self-assembled monolayers
•     For protein adsorption, Interaction forces
  include dipole and induced dipole moments,
  hydrogen bond forces and electrostatic potentials

Schematic representations of several proteins
attached to the substrate that are exposed to
mechanical stress
Thomas E. Fisher, Mariano Carrion-Vazquez,
Andres F. Oberhauser, Hongbin Li, Piotr E.
Marszalek, Julio M. Fernandez. Single Molecular
Force Spectroscopy of Modular Proteins in the
Nervous System Neuron, Vol. 27, Sep. 2000,
435-446
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Tip Functionalization

• Why do we need to functionalize the tip?
• To alter the surface properties of the tip so
  that it gains affinity to attach to the required
  end of the biomolecules
• Helps in selectivity of single molecule

Schematic Representation of Silanization Process

• Methods used for Functionalization
• Plasma Treatment to get desired hydrophobicity
• Silanization
• Using a spacer as a linker molecule in between
  eg. PolyEthyleneGlycol (PEG)
• Using appropriate biomolecules for linking
  antigen or ligand

Different ligands tethered to AFM tip via
flexible PEG linker
C. Tolksdorf, I. Revenko Choosing AFM Probes
for Biological Applications Cordula M. Stroh
et. al. Tools for single molecule Recognition
Force Microscopy and Spectroscopy Molecular
Imaging Application Note
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Tip Parameters
Tip Parameters play an important role for
single-molecule recognition and spectroscopy

• Main Features
• Tip Sharpness (Typically 10 nm)
• Dull tips for some biological applications
  corrections required
• Cantilever Stiffness force measurements
• Q-factor typically 100300 in air and 1 in
  liquid using positive feedback system it can be
  controlled

Sketch of a sensor molecule (brown) tethered to
the AFM tip through a PEG linker
Sharpness of the tip required for biological
applications
Cordula M. Stroh et. al. Tools for single
molecule Recognition Force Microscopy and
Spectroscopy Molecular Imaging Application
Note http//www.lot-oriel.com/uk/htm/all/obe12b
.php
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Applications and Developments

• 1) Cell Adhesion and Rupture
• Contact Formation
• Adhesion
• Rupture

Force Spectroscopy of adhesion between individual
D. discoideum cells

• 2) Chemical Force Spectroscopy on Single-Walled
  Carbon Nanotubes
• Nanotubes are good components for polymeric
  composites
• Using Force Spectroscopy, their affinity to
  various molecules can be found

3) Estimating Bulk Properties using measurements
at Molecular level
Martin Benoit, Daniela Gabriel, Gunther
Gerisch, Hermann E. Gaub. Discrete Interactions
in cell adhesion measured by single-molecule
force spectroscopy Nature Cell Biology, Vol. 2,
June 2000, 313-317
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Typical Force Curve

• The curve shows a typical force characteristics
  of a biomolecule (typically a folded protein)
• Each drop in force (2-3) corresponds to one
  unfold in the molecular structure
• Similar order of peaks show that same force is
  required to unfold it every time
• Number of peaks tell about the total number of
  folds in the molecule

The Forced extension of Modular Proteins exhibits
a Saw-Tooth Pattern
Thomas E. Fisher, Mariano Carrion-Vazquez,
Andres F. Oberhauser, Hongbin Li, Piotr E.
Marszalek, Julio M. Fernandez. Single Molecular
Force Spectroscopy of Modular Proteins in the
Nervous System Neuron, Vol. 27, Sep. 2000,
435-446
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Conclusion

• A very powerful tool for various applications
  like
• Characterizing single biomolecules
• Cell-cell interactions
• Molecular interaction with surfaces
• Challenges involved because of mechano-chemical
  interactions
• Not very well-defined or specific process
  Parameters of functionalization vary with
  different kind of molecules to be analyzed
• Huge potential in upcoming era of Biotechnology

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Thank You Questions ??