1. Mohammed Zuned Desai Michael James Wong Koji Hirota Areio Hashemi Group D

2.  Background  Applications  Description  Objectives  Methodology  Fabrication  Results  Future Work  Gantt Chart  References

3. Introduction  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

4. Advantages of MT  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

5. Applications  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.

6.  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

7. suspension of microspheres molecular layer transparent substrate NSNS CCD objective mirror layer modified with ligands layer modified with protein force Experiment design: Working View 7 Design of Magnetic Tweezers

8. Negative Control: No inhibitor on the surface time F F beads settle magn. wash @ 1 pN beads settle magn. wash @ 1 pN data collection @ 12 pN ° ° ° ° ° ° ° ° ° ° ° ° ° °° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° °° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° °° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° °° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° °° ° ° ° ° ° ° ° ° ° ° ° ° Beads and surfaces coated with Bovine Carbonic anhydrase and sulfonamide inhibitor 8 Dissociation of CA-sulfonamide complexes:

9.  Calibrating design: Side View Square capillary with suspension of microspheres NSNS CCD force 9

10.  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 FdFd FgFg 10 FMFM

11. Objectives  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

12. Methods  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

13.  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

14.  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

15.  Small vs Big Core Iron 0.25 in 0.5 in 1.5 in 0.37 in 0.75 in 1.25 in Iron CoilCore  Small core gave better uniform magnetic gradient

16.  Magnetic field and Magnetic gradient

17.  Tip Shape Angle 161.8 0 76 0 45.2 0 Arc Angle 30 0 45 0 60 0 90 0 Concave 30 0 45 0 60 0 90 0 θ Length Small: 0.01mm Medium: 0.08mm Large: 0.15  Flat showed best results  Second best was tip with angle of 161.8 0

18.  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

19. A) Small double vs Big double B) Small double with Shapes (tip, arc, concave)  180 0 shows best results C) Changing angle (60 0, 90 0,180 0 ) θ θ = 15 0 θ = 45 0 θ = 60 0 2mm

20.  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 Mu Metal 0.25 in 0.5 in 1.5 in

21.  Testing to see how coil manipulation effects the magnetic field  Increasing the number of coils  Location of the coil A) C)B)

22. 70 0

24. Overall Design Light source DC power supply CCD camera Stage Reflect mirror Objective lens Stage adjuster Magnet

25. StageStage Manipulator

26. Magnet Mirror

27.  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)

28. Magnetometer Probe Tip Magnet Adjustments Knobs DC power supply Scotch Tape

29. G vs LengthdG/dL vs Length 1Gauss = 1 x 10 -4 Tesla (B)

30. B vs LengthdB/dL vs Length

31.  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

32.  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: 100 th of a mm  Start working with beads ◦ Velocity measurements ◦ Force measurements

34.  Dr. Valentine Vullev  Dr. Sharad Gupta  Dr. Hyle Park  Dr. Jerome Schultz  Gokul Upadhyayula  Hong Xu

35.  1) Neuman, Keri C, and Nagy, Attila. “Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy.” Nature Publishing Group Vol. 5, NO. 6. June 2008.  2) Danilowicz, Claudia, Greefield, Derek and Prentiss, Mara. “Dissociation of Ligand-Receptor Complexes Using Magnetic Tweezers.” Analytical Chemistry Vol. 77, No. 10. 15 May. 2005.  3) Humphries; David E., Hong; Seok-Cheol, Cozzarelli; Linda A., Pollard; Martin J., Cozzarelli; Nicholas R. “Hybrid magnet devices fro molecule manipulation and small scale high gradient-field applications”. United States Patent and Trademark Office, An Agency of The United States Department of Commerce.. January 6, 2009.  4) Ibrahim, George; Lu, Jyann-Tyng; Peterson, Katie; Vu, Andrew; Gupta, Dr. Sharad; Vullev, Dr. Valentine. “Magnetic Tweezers for Measuring Forces.” University of California Riverside. Bioengineering Senior Design June 2009.  5) Startracks Medical, “Serves Business, Education, Government and Medical Facilities Worldside.” American Solution. Startracks.org, Inc. Copyright 2003.  5) Startracks Medical, “Serves Business, Education, Government and Medical Facilities Worldside.” American Solution. Startracks.org, Inc. Copyright 2003.