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Mohammed Zuned DesaiMichael James Wong
Koji HirotaAreio Hashemi
Group D
Background Applications Description Objectives Methodology Fabrication Results Future Work Gantt Chart References
What are Magnetic Tweezers (MT)?◦ Scientific instrument used for studying molecular
and cellular interactions◦ Ability to apply known forces on paramagnetic
particles using a magnetic field gradient◦ One of the most commonly used force
spectroscopy techniques Atomic Force Microscopy Optical Tweezers
They do not have problems of sample heating and photodamage that effects optical tweezers
Magnetic forces are orthogonal to biological interactions
Offer the prospect of highly parallel single-molecule measurements ◦ Hard to achieve with other single-molecule
force spectroscopy techniques
The magnet configurations are relatively easy to assemble
Ideally suited for the study of DNA topology and topoisomerases
Study Molecular interactions 65pN to rupture bond between lectin and RBC membrane-
bound glycolipids. 60-130pN to extract beta2-integrins (CD18) from neutrophil
membrane in 1-4sec 100pN to extract integral glycoprotein from cell lipid bilayer
(RBC membrane) 165pN to rupture P-selectin bond with leukocyte-membrane-
bound P-selectin glycoprotein ligand-1. 40-400pN to separate a pair of cell adhesion proteoglycan
molecules on marine sponge cell surfaces.
How do magnetic tweezers work?
http://www.biotec.tu-dresden.de/cms/fileadmin/research/biophysics/practical_handouts/magnetictweezers.pdf
Aspects:• Two magnets• Magnetic Field• Magnetic Gradient• Superparamagnetic
beads• Surface Molecules
suspension of microspheres
molecular layer
transparent substrate
N
S
CCD
objective
mirrorlayer modified
with ligands
layer modified with protein
force
• Experiment design: Working View
7
Design of Magnetic Design of Magnetic TweezersTweezers
Negative Control:No inhibitor on the surface
time
F
time
F
beadssettle
magn. wash @ 1 pN
beadssettle
magn. wash @ 1 pN
data collection @ 12 pN
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Beads and surfaces coated with BovineCarbonic anhydrase and sulfonamide inhibitor
8
Dissociation of CA-sulfonamide Dissociation of CA-sulfonamide complexes:complexes:
Calibrating design: Side View
Square capillary with
suspension of microspheres
N
S
CCD
force
9
force
Force calculations using Stoke’s drag equation:◦ Calibrate:
Distance between the core of the electromagnet and paramagnetic beads
Current flowing through the coil of the magnet
Example: ◦ Time it takes bead to move vertically 0.5mm =
3.46s◦ Velocity of bead (v) = 0.1445 mm/s◦ Fluid’s viscosity (u)= 0.998 mPa s◦ Radius of bead (r) = 1.5 um◦ Drag Force = 4.07379 pN
Gravitational Force ~ 0.3 pN
rFd 6
Fd Fg
10
FM
gdM FFF
Design and fabricate magnetic tweezers that is capable of achieving forces up to 100pN◦ Current design can achieve 2pN◦ Consist of a single magnet
Introduce illumination for bright-field transmission microscopy
Using Finite Element Method Magnetics (FEMM) to predict the geometries of the magnet and that will produce the largest possible field gradients
Machine and assemble the design that will produce the largest field gradients
Calibrate the magnet so it is ready for data acquisition
Open source finite element analysis software package for solving electromagnetic problems.
Good for processing:◦ 2D planar and Axisymmetric problems ◦ Magnet◦ Electrostatic ◦ Heat and Current Flow
It is a simple, accurate, and low computational cost freeware product, popular in science and engineering.
Reliability comparable to commercial software Referenced in several Journals Used by several reputable societies
IEEE Magnetics UK and Japan Magnetics
A) Characteristics of Magnets Core size Tip shape
B) Double Magnet Runs Test FEMM reliability Core, Shape, and Angle
C) Core Material Mu metal
D) Coil Manipulation Increasing the number of coils Changing their location
Looking at how these characteristics affect the magnetic gradient
¼ inch
1.5 inch
1/8 inch
Small vs Big Core
Iron
0.25 in
0.5 in
1.5 in
0.37 in
0.75 in
1.25 inIron
Coil Core
Small core gave better uniform magnetic gradient
Magnetic field and Magnetic gradient
Tip Shape
Angle161.80
760
45.20
Arc Angle300
450
600
900
Concave300
450
600
900
θ
LengthSmall: 0.01mmMedium: 0.08mmLarge: 0.15
Flat showed best results Second best was tip with angle of 161.80
Whatever characteristics of single magnet we don’t want to blindly assume are the same for double magnets◦ Ex: Flat small has better magnetic gradient but
this does not mean that Flat small gives better gradient with double magnets so we run double magnets
Reliability of FEMM through comparison of single and double results
A) Small double vs Big double
B) Small double with Shapes (tip, arc, concave)
1800 shows best results
C) Changing angle (600, 900,1800)
θ
θ = 150
θ = 450
θ = 600
2mm
Mu Metal vs Iron Different tip shapes Double vs Single
Angle Tips
The Small Mu Metal flat magnet showed the best results in single and double magnet runs
MuMetal
0.25 in
0.5 in
1.5 in
Testing to see how coil manipulation effects the magnetic field
Increasing the number of coils Location of the coil
A) C)B)
700
Overall Design
Light source
DC power supply
CCD camera
Stage
Reflect mirror
Objective lens
Stage adjuster
Magnet
Stage Stage Manipulator
MagnetMirror
Objective: Verify that flat tip shows the best results Prove that the tip gives the largest magnetic field gradient
values at very short distances.
Tested different tips Flat Cylinder Tip
Parameters◦ Voltage: 3v, 6v, 12v◦ Current: 0.1 Amps◦ Distance:
0-.5mm (0.1mm increments) .5-3.1mm (0.2mm increments)
Magnetometer Probe
Tip
Magnet
AdjustmentsKnobs
DC power supply
ScotchTape
G vs Length dG/dL vs Length
1Gauss = 1 x 10-4 Tesla (B)
B vs Length dB/dL vs Length
Finished experimenting on magnet characteristics to obtain greatest magnetic field gradient.
Fabricated majority of the device setup
Performed trial runs on single magnet with different tips to verify certain trends
Ship final magnetic design with the material to the Robert M. Hadley Company.
Locate homogeneous field Experiment with horizontal distance with very small
increments Capability: 100th of a mm
Start working with beads◦ Velocity measurements◦ Force measurements
Dr. Valentine Vullev Dr. Sharad Gupta Dr. Hyle Park Dr. Jerome Schultz Gokul Upadhyayula Hong Xu
1) Neuman, Keri C, and Nagy, Attila. “Single-molecule force spectroscopy: 1) Neuman, Keri C, and Nagy, Attila. “Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy.” optical tweezers, magnetic tweezers and atomic force microscopy.” Nature Nature Publishing GroupPublishing Group Vol. 5, NO. 6. June 2008. Vol. 5, NO. 6. June 2008.
2) Danilowicz, Claudia, Greefield, Derek and Prentiss, Mara. “Dissociation of 2) Danilowicz, Claudia, Greefield, Derek and Prentiss, Mara. “Dissociation of Ligand-Receptor Complexes Using Magnetic Tweezers.” Ligand-Receptor Complexes Using Magnetic Tweezers.” Analytical ChemistryAnalytical Chemistry Vol. 77, No. 10. 15 May. 2005.Vol. 77, No. 10. 15 May. 2005.
3) Humphries; David E., Hong; Seok-Cheol, Cozzarelli; Linda A., Pollard; Martin 3) Humphries; David E., Hong; Seok-Cheol, Cozzarelli; Linda A., Pollard; Martin J., Cozzarelli; Nicholas R. “Hybrid magnet devices fro molecule manipulation J., Cozzarelli; Nicholas R. “Hybrid magnet devices fro molecule manipulation and small scale high gradient-field applications”. United States Patent and and small scale high gradient-field applications”. United States Patent and Trademark Office, An Agency of The United States Department of Commerce. Trademark Office, An Agency of The United States Department of Commerce. <http://patft.uspto.gov>. January 6, 2009. <http://patft.uspto.gov>. January 6, 2009.
4) Ibrahim, George; Lu, Jyann-Tyng; Peterson, Katie; Vu, Andrew; Gupta, Dr. 4) Ibrahim, George; Lu, Jyann-Tyng; Peterson, Katie; Vu, Andrew; Gupta, Dr. Sharad; Vullev, Dr. Valentine. “Magnetic Tweezers for Measuring Forces.” Sharad; Vullev, Dr. Valentine. “Magnetic Tweezers for Measuring Forces.” University of California Riverside. Bioengineering Senior Design June 2009.University of California Riverside. Bioengineering Senior Design June 2009.
5) Startracks Medical, “Serves Business, Education, Government and Medical 5) Startracks Medical, “Serves Business, Education, Government and Medical Facilities Worldside.” American Solution. Startracks.org, Inc. CopyrightFacilities Worldside.” American Solution. Startracks.org, Inc. Copyright 2003. <http://images.google.com/imgres?imgurl=http://www.startracksmedical.com/supplies/invertedmicroscope.jpg&imgrefurl=http://www.startracksmedical.com/supplies.html&usg=__butCY2zWJa7nAkwkjiPxX_mFy0=&h=450&w=450&sz=24&hl=en&start=2&um=1&tbnid=XH6gnQuJLS7bRM:&tbnh=127&tbnw=127&prev=/images%3Fq%3Dinverted%2Bmicroscope%26hl%3Den%26sa%3DN%26um%3D1>
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