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>