Design of Magnetic Tweezers Team D Mohammed Zuned Desai Areio Barzan Hashemi Koji Hirota Michael James Wong

Magnetic Tweezers Exerts magnetic forces to determine mechanical properties of molecules, proteins, chemical bonds Advantages The magnet configurations are relatively easy to assemble Magnetic forces are orthogonal to biological interactions Offer the prospect for massively parallel single-molecule measurements Frame better, what last years group had ( better field stregnth and gradiest) Improve the magnet (better coiling) Improve the configuration (the whole two magnet and machinging the tip) 1st show how twe are implemented into the larger setup Figure 1: Illustration of Magnetic Tweezers, adapted from http://www.nature.com/nature/journal/v421/n6921/images/nature01405-f2.0.jpg

How do Magnetic Tweezers Work? Magnet(s) Stage PDMS Wells Containing Sample 10x Objective Lens CCD Camera Light Source Mirror layer modified with protein force Magnetic tweezers is essentially and inverted microscope (with the objective on the bottom), and a magnet on top. We have a CCD camera in order to observe the interactions between protein-ligand complexes, where proteins are coated on the super paramagnetic beads and glass surface is coated with the ligand By turning on the electromagnet, we can exert specific force on these beads. At small forces, beads that did not complex with the surfaces are washed And at higher forces, we are able to observe the dissociation of the protein-ligand complex -remover caplirry put slide with holes Incorporate the force and calibrations (paramagentic beads and force felt by beads are equal to m del b) layer modified with ligands Figure 2: Diagram of setup of magnetic tweezers Figure 3: Animation of how magnetic tweezers work 3

How Magnetic Tweezers Work? What do these forces depend on? The force on the paramagnetic beads depend on the magnetic moment and the magnetic field gradient ( ) To achieve higher forces we either increase or m Force Calculations: -**Later on we will calculate the force by on obtaining the rersulting terminal velocity which is a resultant of del B FM Fd Fg

Magnetic Field Gradient What do we mean by magnetic field gradient ? Want to achieve a high gradient For the same distance we want a constant change in gradient 0.2 mm B = 800G B = 700G B = 620G B = 560G Figure 4: Diagram demonstrating the definition of homogeneous gradient Gauss Need arieos equ Show how ma -this is del b Sample attached to paramagnetic beads

Objectives Main goal is to focus on attaining forces with the Magnetic Tweezers for single-molecule measurements (e.g. 5 – 100 pN): Design producing the highest gradient Achieving force higher than 1.5pN (previous group) Calibrating the selected design Improve the magnetic gradient Figure 5 and 6: Setup of last years senior design group taken at different angles.

High Magnetic Gradient To maximize the magnetic gradient Build the stronger magnet(s) with materials Geometry shape and position Using FEMM (Finite Element Method Magnetics) Figure 8: Double Magnet FEMM Design Figure 9: Single Magnet FEMM Design

Single Magnet Design Using FEMM simulation program Choose the materials Design the core including length, diameter and shape Outcome CORE Steel 127mm (5 in.) core length 6.35mm (0.25 in.) core diameter Flat Shape COIL 30 Gauge Copper wire 2500 coil turns 91.44mm (3.6 in.) coil length 15.49mm (0.61 in.) coil diameter Figure 11: Picture of the magnet (coil and core)

Double Magnets Design From the literature research and FEMM simulation, this design of double magnets should exert higher magnetic gradient Figure 10: Drawing of final double magnet design

Magnetic Tweezers Setup Single Magnet Up to 5.9 pN Double magnets Up to 3.5 pN Inverse relationship of strength of magnetic field over distance Figure 12: Close up picture of single magnet design Figure 13: Close up picture of double magnet design

Measuring Magnetic Gradient by Gaussmeter Measure magnetic gradient over distance Graphs to be linear Magnetic gradient is decreasing at a constant rate Compare magnetic gradient within working distance (2mm) Single Magnet: 148.43 G/mm Double Magnet: 120.56 G/mm Mike is Figure 14 and 15: Comparison of the gradient results for the single magnet and double magnet

Calibration Process: Setup **-How is the capillary tube related to the first layered slide. -Previous used a slide with pdms, this slide uses capillary tube with beads resting on the surface due to gravity. We need to do this calibration so that we know what forces are exerted at certain distances. Only for calibration so don’t need coat -camera location side=calibration, bottom=pdms Figure 16: Calibration setup for the single magnet Figure 17: Calibration setup for the double magnet

Actual Setup Adjustable Stage Magnet(s) CCD Camera 10x Objective Lens Sample Stage with Capillary Tube LED Light Source Power Supply Figure 7: Picture of the setup of our final design

Calibration Process 1) Zero apparatus Have the tip of the magnet close to the capillary tube 2)Inject beads into the capillary tube 3)Turn on the power source 4) Note time it takes for the bead to travel 0.5mm

Calibrating Magnetic Tweezers Force calculations using Stoke’s drag equation: Calibrate: Distance between the core of the electromagnet and paramagnetic beads Gravitational Force ~ 0.3 pN Example: Time it takes bead to move vertically 0.5mm = 9.4s Velocity of bead (v) = 0.054 mm/s Fluid’s viscosity (u)= 3.63 mPa s (40% Glycerol Solution) Radius of bead (r) = 1.5 um Net Force (Fm) = 5.91 pN FM Fd Fg To use magnetic tweezers, we need to calibrate two parameter: First, the distance between the core of the electromagent and the force exerted on the paramagnetic beads… and Second, relation between the current flowing through the coil of the electromagnet and the force exerted on the beads We can do this by using stoke’s law Once we let the beads settle and then apply a certain voltage at a specific magnet height, we can determine the drag force. We can determine the terminal velocity by measuring the time it takes the bead to move vertically for a certain distance By plugging in the velocity, fluid’s viscosity and radius of the bead, we can determine the drag force. By measuring the time it takes the beads to move down in the absence of external force, we calculated the gravitational force to be ~0.3pN -need duplicate simplified slide in the beginning -Increase the Force not FIED… Remember the force equation of a solonoid, increase the force want del cross b to be as higest possible (we want the highest gradient which inturn implies the highest velocity) ***Velcity that we obtain is a resultant of the gradient acting on the bead force 15 Figure 18: Animation of forces acting on the bead 15

Sample Calibration Video 1mm 0.75mm 0.5mm Constant velocity questions 2 mega pixels 0.25mm 0mm Video 1 : Sample video of beads moving for the calibration process

Calibration Results 3V Results Single Magnet Calibration Force at 1mm: 1.02pN Force at 2mm: 0.98pN Double Magnet Calibration Force at 1mm: 0.76pN Force at 2mm: 0.73pN 6V Results Single Magnet Calibration Force at 1mm: 2.23pN Force at 2mm: 1.85pN Double Magnet Calibration Force at 1mm: 1.44pN Force at 2mm: 1.20pN -** Interested in 0.2mm because that is the working distance of the pdms slides. Figure 19 and 20: Caparison of the forces the single and double magnet could achieve using 3V and 6V

Calibration Results 12V Results Single Magnet Calibration Force at 1mm: 5.91pN Force at 2mm: 4.84pN Double Magnet Calibration Force at 1mm: 3.52pN Force at 2mm: 3.18pN -**By increasing the votlage we increase the current from (0 to 0.4A) which leads to an increase in the force As you increase the voltage you increase the current (which increase the force) (the field depends on the current) 2mm, bc of wells Figure 21: Caparison of the forces the single and double magnet could achieve using 12V We are mainly concerned about the 12V measurements The results show that the single magnet can achieve higher forces than the double magnet

Conclusion We accomplished our objectives: 1) We were able to design and build a pair of magnetic tweezers that can achieve over 1.5pN Single magnet magnetic tweezers can achieve 3.94times more force than old design Double magnet magnetic tweezers can achieve 2.35times more force than old design 2) Successfully able to calibrate both magnetic setups

Future Work m = magnetic moment in a superparamagnetic bead B = magnetic field in Tesla I = amperes n = turns per meter K = permeability = magnetic constant Permeability of steel = 100 Permeability of Mu Metal = 20,000

Future Work Mu Metal Permeability Heat treatment Nickel-iron alloy Ability to support magnetism 200 times than that of steel Heat treatment Reduces amount of oxygen in metal Gains back permeability that was lost

Future Work Heat Conduction Thermoelectric Cooling Peltier Cells Liquid Cooling System Water Blocks

Future Work Stage Holds PDMS wells and tube Repeatable parameters Detachable Fitted to optics table or microscope if needed

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

References 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. <http://patft.uspto.gov>. 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. <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> 6) Janshoff A, Neitzert M, Oberdorfer Y, Fuchs H. Force spectroscopy of molecular systems-single molecule spectroscopy of polymers and biomolecules. Angew Chem Int Ed 2000;39:3212-3237.

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