Magnetic Tweezers for Accurate Single-Molecule Force Measurements Noah Johnson BRITE 2009.

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Presentation transcript:

Magnetic Tweezers for Accurate Single-Molecule Force Measurements Noah Johnson BRITE 2009

Aims Design and calibration Eliminate non-specific interactions Study protein-ligand model systems

What are magnetic tweezers? Tiny forces: pN Single-molecule measurements Optical Tweezers AFM

Advantages of magnetic tweezers Record single molecule events in parallel Don’t interfere with biological interactions Requires low energy Maintains constant force

Design and calibration F M = F d + F g F d =6π ηr v FgFg FMFM FdFd N CCD 1mm square glass capillary with bead suspension 10x objective 12V electromagnet CCD camera S

Gravitational force FgFg The beads are polystyrene with a broken magnetite core ρ Magnetite = 3.3 g/cm 3 ρ Polystyrene = 1.05 g/cm 3 ρ Beads = 1.9 g/cm 3 F g = mg = ρ beads V fluid g = 0.3pN

Bead clumping in water

Single beads with TWEEN 1.33mM TWEEN 20 in 10X PBS

Eliminating non-specific interactions distance glass 11 nm ~ 3 PEG molecules / nm 2 Need at least 20nm separation! Wan, Thomas, Guthrie &Vullev,Annals of Biomed Eng, 2009, 6,

Surface derivatization glass silanization deprotection reductive amination amide coupling protein immobilization glass NH protein glass Wan, Thomas, Guthrie &Vullev,Annals of Biomed Eng, 2009, 6,

Contact angles Validate surface chemistry PEG ± 0.48° Clean glass-26.7 ± 0.24°

Bead Derivatization DIC 1,3-Diisopropylcarbodiimide HOBt 1-Hydroxybenzotriazole 13

Eliminating non-specific interactions PDMS well filled with magnetic bead suspension molecular layer glass slide NSNS CCD 25x objective mirror

Expected results ° ° ° ° ° ° ° ° ° ° ° ° ° °° ° ° ° ° ° ° ° ° ° ° ° ° ° ° time F beads settle magnetic wash <1 pN NSNS

Our results  PEGylated surface  PEGylated beads

Our results  PEGylated surface  PEGylated beads

Our results 91.3%73.3% 47.2%26.6% Substrate Beads PEG No PEG  Percentage of beads removed by a 0.357pN magnetic wash

Progress  Calibration and surface chemistry  Non-specific interactions experiments  Protein-ligand dissociation kinetics and trajectories

Future Work Add proteins/ligands to PEG Study dissociation kinetics and trajectories glass bead substrate

Acknowledgements Gokul Upadhyayula Dr. Sharad Gupta Stephen Bishop Sean Guthrie Dr. Valentine Vullev Jun Wang

Carbonic anhydrase Test magnetic tweezers Well characterized system Determine k d and compare Magnet Stage Objective 10x MirrorCCD