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THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE

DEPARTMENT OF BIOMEDICAL ENGINEERING

A CONTROL STUDY USING DNA TO TEST THE EFFECTS OF SHEAR STRESS ON VON WILLEBRAND FACTOR

MAYA ANN-MARIE JANKOWSKA SPRING 2017

A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biomedical Engineering with honors in Biomedical Engineering

Reviewed and approved* by the following:

Keefe B. Manning Associate Professor of Biomedical Engineering Schreyer Associate Dean of Academic Affairs Thesis Supervisor

William O. Hancock Professor of Biomedical Engineering Honors Advisor

Peter J. Butler Engineering Associate Dean for Education Professor of Biomedical Engineering Faculty Reader

* Signatures are on file in the Schreyer Honors College. i

ABSTRACT

In an effort to understand how the von Willebrand Factor (vWF), a blood clotting glycoprotein, and applied shear forces are related, this project focuses on a positive control study using plasmid DNA. Elevated shear stress levels, commonly introduced after ventricular assist device (VAD) implantation, are believed to cause unfurling and subsequent enzymatic cleavage of vWF. This makes vWF impotent and subjects the patient to high risks of bleeding. However, due to vWF's large and complex structure, a positive control of plasmid DNA will produce a more predictable experiment. Through attaching enzymatically-cut and treated plasmids around the entire exterior of biotinylated polystyrene beads, an effective radius of the bead plus the

DNA length is assumed. The hypothesis is the effective radius of DNA is independent of shear rates. Within this study, the aims are to (1) determine 2 plasmid-enzyme pairs with one of comparable length to unfurled vWF and one of shorter length, (2) attach the treated enzymatically-cut plasmid to beads, (3) subject the beads to shear forces in an optical trap to compare effective radii of DNA-coated beads to non-coated beads, and (4) determine the effect of shear rates on DNA-coated and non-coated bead effective radii. Thus, the control study post- processing techniques and data can be reproduced and applied to vWF. A quantifiable difference in bead effective radius is observed between the DNA-coated beads and the non-coated beads.

This presumably confirms that DNA was coated onto the beads. Effective radii was not significantly affected by shear rates when comparing the beads on each slide. ii

TABLE OF CONTENTS

LIST OF FIGURES ...... iii

LIST OF TABLES ...... iv

ACKNOWLEDGEMENTS ...... v

Chapter 1 Introduction ...... 1

Von Willebrand Factor ...... 2 Von Willebrand Disease ...... 6 Shear Stress Fluid Mechanics Theory ...... 8 Ventricular Assist Devices ...... 11

Chapter 2 Optical Trap Theory ...... 14

Chapter 3 Parallels between DNA and vWF ...... 17

Effective Radius ...... 18 Objectives and Hypothesis ...... 19

Chapter 4 Materials and Methods ...... 20

Preparing the DNA-Coated Beads ...... 20 Running the Bead on the Optical Trap ...... 26

Chapter 5 Results ...... 38

Spring Constant ...... 38 Bead Displacement...... 41 Effective Radius Values ...... 43

Chapter 6 Discussion ...... 47

DNA-Coated Beads Analysis ...... 47 Optical Trap Analysis ...... 50

Chapter 7 Future Work ...... 55

Appendix A Displacement and Phase Raw Data - pACYC177 ...... 58

Appendix B Displacement and Phase Raw Data – pUC19...... 59

BIBLIOGRAPHY ...... 60 iii

LIST OF FIGURES

Figure 1: Elongation of vWF under high shear (a) cartoon depiction of vWF below and above critical shear rate (b) fluorescence image of the relaxation of vWF after reducing shear rate to 0 s-1; adapted from Siediecki et al.8 ...... 2

Figure 2: vWF facilitating the adhesion between exposed collagen and its platelet receptors (GPIb/IX) to assist blood clotting at wound site, adapted from Geisen et al.1 ...... 4

Figure 3: Graphic representation of vWF A-D domains and main binding sites, including ADAMTS13 cleavage site. Regions where mutations in types of VWD have been found are also indicated; adapted from De Meyer et al.15 ...... 5

Figure 4: (a) A healthy aortic valve in comparison to a severely stenotic valve; adapted from Medtronic19 (b) the decrease in high-molecular weight (ultra-large) vWF with increasing pressure gradient. Increasing pressure gradient is directly correlated with increasing valve stenosis; adapted from Vincentelli et al.18 ...... 7

Figure 5: Laminar, or smooth fluid movement in orderly layers, Couette flow between two parallel plates with a stationary lower plate and an upper plate moving at some arbitrary velocity; adapted from Cengel et al.22 ...... 9

Figure 6: Electrophoresis of vWF multimers in a healthy and VAD-implant patient, adapted from Geisen, et al.1 Smaller multimers can be seen lower in the gel as they pass faster through the gel. It is evident that the VAD patient is missing large vWF bands in the top portion of the gel. The densitometry curve on the right indicates this phenomenon...... 12

Figure 7: Correlation between increasing VAD operating speed and decreasing percentage of high molecular weight vWF multimers; adapted from Meyer et al.24 ...... 13

Figure 8: Optical trap representation with bead. Beads are attracted to the center of the beam and slightly above the beam waist. Hooke’s law is at play with the spring force in the trap; adapted from Candela26 ...... 15

Figure 9: A single beam trap; adapted from Svoboda et al.27 The light focus is seen at the top indicated by the darkest region. The black arrows indicate the light focusing through the circular bead. The gray arrows pointing relatively upwards indicate reaction forces pulling the bead upwards...... 16

Figure 10: Cartoon depiction of “effective radius” extending from the center of the bead to the edge of the DNA/vWF. DNA effective radii will resemble the ‘r3’ bead, adapted from Corsetti.31 ...... 18

Figure 11: Depiction of plasmid with multi-cloning site (MCS), a DNA segment with several unique sites for restriction enzymes to cut. These restriction sites are not present anywhere else on the plasmid...... 21

Figure 12: Cartoon depiction of cut plasmid with biotinylated 3’ ends ...... 23 iv

Figure 13: Flow chamber sealed on all four sides with the right chamber containing control beads and the left chamber containing DNA-coated beads. Two slides were constructed for the 2 DNA-coated bead groups...... 27

Figure 14: The optical trap consists of the IX71 Olympus inverted microscope with QPD, condenser, objective, stage, laser, and CCD camera...... 29

Figure 15: Arroyo Instruments 6310 ComboSource Laser Diode controller set to 540 mAmps. 29

Figure 16: QPD LabView program live preview with QPDsum (green line) maximized and QPDx and QPDy (red and white lines). QPD x and y are slightly separated to display the two lines, for clarity...... 31

Figure 17: The LabView Spring Constant program showing a sinusoidal 1 V wave applied to the stage/bead and the resulting spring constant displayed above the wave. A minimum of 5 beads were tested for each slide’s spring constant calculations...... 32

Figure 18: Triangular waveform used in stage oscillation in pre-shear program, adapted from Corsetti.31 ...... 33

Figure 19: LabView Pre-shear Program, with the triangular stage oscillation, as read by the QPD. The arrows denote the transition from stage oscillation in the x-direction to the y-direction, which was created by maximizing the QPDsum in the QPD calibration described earlier. All pre-shear for the experiments was in the x-direction...... 35

Figure 20: LabView Program for data collection, set to sine wave with 1 V amplitude stage motion. The red line shows the stage motion and the white line shows the phase of the sinusoidal motion of the bead...... 36

Figure 21: Camware Program showing a CCD image of a trapped bead undergoing data collection. The center axis can be used as a reference point to see that the bead is slightly displacing during data collection...... 37

Figure 22: Each bead trial and its corresponding spring constant, per slide. Lines of best fit and R2 values are shown...... 40

Figure 23: Spring constant distributions for both slides shown in a) box plot and b) dot plot form. pACYC177 spring constants (n=9) and pUC19 spring constants (n=5) ...... 41

Figure 24: Displacement (V) values of the bead from the center of the trap. (A) pACYC177 slide (B) pUC19 slide. Standard error of the mean bars are shown...... 42

Figure 25: Effective radius values (µm) for (a) pACYC177 slide (b) pUC19 slide. Each slide graph shows points for control beads and DNA-coated beads at low and high shear rates. Standard error of the mean bars are shown...... 45

Figure 26: CCD image showing three different sized non-coated beads, demonstrating the variability in the manufacturer’s bead size ...... 51 v

LIST OF TABLES

Table 1: Input Amplitude and Frequency with the corresponding stage velocities and shear rates34

Table 2: Spring Constants and Conversion Factors for both DNA-coated bead slides ...... 38

ACKNOWLEDGEMENTS

First and foremost, I would like to thank Dr. Manning as my thesis research advisor. His patience and support was vital for my completion of the thesis. His suggestions allowed me to connect with many people in the biomedical engineering network at Penn State, which was extremely beneficial. Additionally, the time, funds, and resources he provided have been crucial for me to gain this experience and I am very grateful. I would also like to thank Dr. Peter J.

Butler, our optical trap developer, and Dr. William O. Hancock, DNA connoisseur, as my faculty readers for their input and enthusiasm for the project. Their offer to assist in any way was greatly appreciated. Additionally, Joshua Riley and Dave Arginteanu have been crucial to the completion of this thesis by donating their time and creating a foundation for my project. Josh’s assistance and training on the optical trap provided continuing insights into the project. Dave’s expertise in molecular biology was greatly appreciated as I would not have successfully created my DNA-coated beads without him. Their openness to assisting me over the course of the project was valued, acting as a well of answers and knowledge for the project.

I’d also like to thank Xavier Candela’s and Monica Corsetti’s thesis and other parent study contributors. While I did not have the opportunity to work with Xavier and Monica, their theses and time contributions to the lab and research are appreciated and acknowledged.

Lastly, I’d like to thank my mom and dad and Justin for their unending support in me completing this thesis. Their belief in me helped me turn stress into success. 1

Chapter 1

Introduction

Acquired von Willebrand Syndrome (AVWS) is a disease that causes abnormal and spontaneous bleeding into the skin and soft tissues due to malfunctioning von Willebrand Factor

(vWF). The disease slows the blood clotting process, causing protracted bleeding from injuries.

While the parent disease, von Willebrand Disease (VWD), can be inherited, it has been found that a syndrome can also be acquired from the implantation of ventricular assist devices

(VADs).1-4 These devices introduce elevated shear rates into the bloodstream, which cause the unfurling of vWF, or unrolling from its folded state, rendering it impotent.5 There is uncertainty in literature regarding the specific shear rate that induces conformational change. One study found values greater than 2000 s-1 with exposure times of less than 12 seconds have been linked to the unfurling conformational change.6 Another study found that any shear rates above 5000 s-1 leads to conformational changes from the vWF compact configuration.7 Yet another study found shear rates of 3100 s-1 in vitro in platelet-rich plasma (PRP) induced a change.8 Regardless of the shear rate, it is undisputed that shear rates cause a conformational change in vWF. Figure 1 shows a cartoon depiction and fluorescence image of the dynamic conformational change of vWF under shear. 2

Elucidating the threshold shear rates for alteration against the structure and function of von Willebrand Factor would allow implanted medical device inventers to cater their products to avoid these shear values. The parent study aims to reveal the response of shear stress in the bloodstream and its effect on vWF.

Von Willebrand Factor

Von Willebrand Factor is a blood glycoprotein involved in hemostasis and carries procoagulant Factor VIII. Additionally, it is one of the few proteins that carries the ABO blood group antigens. The protein is generated and stored in one of two places: either synthesized and 3 stored in the endothelium in its ultra-large form, or synthesized in megakaryocytes and stored in plasma platelets.9 Specifically, the Weibel-Palade bodies house the protein in endothelial cells and the α-granules of platelets carry the protein through circulation.10 When the vWF is released into the plasma, it circulates as a very large protein, the largest found in plasma.11, 12 The multimer is composed of approximately 2050 amino acids and massed at up to 20,000 kDa.1 This breaks down to 80 subunits of 250 kDa each, linked with disulfide bonds. Once its release has been stimulated, the ultra-large form is hyperactive in binding the platelet receptor, GPIb-IX-V complex, resulting in spontaneous platelet aggregation to stop a wound. vWF then forms ultra- large string-like structures that tether it to the cell wall surface. High shear rates (>5000 s-1), seen in arterial circulation, expose the A1 domain of vWF allowing GPIbα to bind and ultimately permitting vWF to tether platelets.

Within its functionality in blood coagulation, the glycoprotein binds to other proteins, particularly Factor VIII, as mentioned. Factor VIII circulates in plasma bound to inactive vWF in a noncovalent protein complex. Factor VIII is released into the bloodstream during thrombosis, or local blood clotting, and initiates vWF binding to collagen. Collagen, in the vessel endothelial wall, is exposed in a wound and the vWF binds to it and acts as an adhesive with platelets, as seen in Figure 2. 4

Figure 2: vWF facilitating the adhesion between exposed collagen and its platelet receptors (GPIb/IX) to assist blood clotting at wound site, adapted from Geisen et al.1

Other blood proteins and vWF binding site partners include the A3 domain, containing the primary binding site for collagen types I and III, and the A1 domain, containing a binding site for collagen VI and the previously mentioned GPIbα (the primary platelet vWF receptor), seen in Figure 3.5 As a complex and large multimer, vWF has many binding sites and a high efficiency for adhering to collagen and GPIbα, thus assisting to mitigate bleeding.

The multimers can be further processed by proteolysis with metalloproteinase ADAMTS-

13, in the A2 domain. For example, when the glycoprotein is subjected to shear flow, the protein unfurls and the ADAMTS-13 binding site is exposed. The floating ADAMTS-13 plasma protease can cleave in this region, between the AA residues tyrosine 1605 and methionine 1606 in the VWF protein.13 The regulation of this cleavage is vital to maintaining the delicate balance between hemostasis and thrombosis. vWF even exemplifies a unique fold that selectively exposes the ADAMTS-13 proteolysis site for cleavage while keeping the remainder of the A2 domain intact and function.14 However, prolonged exposure of the ADAMTS-13 binding site 5 destroys the balance and cuts the glycoprotein prematurely, making vWF inert within the bloodstream. This reiterates that fact that elevated shear rates are correlated with the unfurling and deterioration of vWF.

One of vWF's characteristic folds involves the self-association of the A1 domain with the

D’D3 and the A2 domain.5 The interactions at this region usually assist stabilizing the coiled state of vWF. However, once blood flow shear stress reaches approximately 2 - 2.5 Pa, the interdomain interaction destabilizes the fold and initiates vWF extension.5 This suggests that other interdomain interactions and folds may each possess their own unique threshold strength, which can be surpassed with increasing shear stress. This would lead to an increased propensity for vWF extension and, if prolonged, ultimate destruction.

6 In summary, within regions of high shear flow, vWF is highly susceptible to unfurling from its highly coiled, quiescent form.5 The coiled conformation blocks the GPIbα and

ADAMTS-13 binding sites during low shear and low velocity in blood flow. vWF is prevented from activating and enlisting platelets and, ultimately, from ADAMTS-13 cleaving it. Once vWF is enlisted to tether to collagen, shown in Figure 2, thrombogenic factors at the wound site lead to unfurling.

Von Willebrand Disease

Von Willebrand Disease (VWD) is the most common hereditary human blood-clotting disease and can be acquired in rarer occurences.10 The disease is characterized by a deficiency or dysfunction of vWF. The diagnosis can be determined by excessive mucocutaneous bleeding, abnormal vWF laboratory studies, and/or family history.10 These patients often have symptoms similar to anemics and are susceptible to bruising and bleeding from the gum and trivial wounds.

Due to the similarity of symptoms to hemophilia and other blood disorders, the disease diagnosis can often be delayed. The most indicative test is usually a laboratory study of vWF and Factor

VIII levels, however, there are varying levels of success across different subtypes of the disease.

There are three subtypes of the disease: type 1 VWD, a partial quantitative deficiency of normal vWF; type 2 VWD, a qualitative deficiency of abnormal vWF; and type 3 VWD, the absence of the vWF protein.16

Type 1 VWD is the most difficult to diagnose because it is marked by partial quantitative deficiencies of vWF. Individuals can have low vWF values, yet be healthy or have a milder version of the subtype. Currently, it is suggested that a vWF deficiency of 15% or less is 7 indicative of Type 1. Although there are several genetic variations that cause this subtype, the genetic variation most suggestive is an AA substitution of tyrosine for cysteine at codon 1584.

However, this substitution is present in 10-20% of patients.17 This indicates the difficulty in diagnosing Type 1 VWD.

Type 2 VWD is characterized by qualitative vWF defects that can either be hereditary or acquired (AVWS). Individuals have higher reliability in testing positive for the disease than

Type 1. Several studies have found other physiological conditions correlated with the propagation, or acquirement, of Type 2 VWD. One of these studies found a significant correlation between severe aortic stenosis and Type 2 VWD.18 Figure 4(a) shows a severely stenotic aortic valve in comparison to a normal valve. Figure 4(b) shows the studies finding that for patients with severe stenosis, indicated by an increasing mean transvalvular pressure gradient, the percentage of ultra-large vWF decreased from 12% to 6%.18 Ultra-large vWF is the inactivate state of vWF. a) b)

8 The stenotic regions in the aorta experience shear flow, which proliferates structural changes of vWF, thus introducing AVWS. Another study found all continuous-flow left ventricular assist device (LVAD) recipients had acquired AVWS after their LVAD placement.20

Although VADs are largely successful, bleeding events are a primary complication for sustained

VAD use, linking the devices to VWD. Of particular interest to this study is acquired Type 2

VWD, or AVWS, due to its symbiotic acquirement with LVADs.

The most severe type of the disease is Type 3 VWD marked by virtually complete deficiency of vWF. The autosomal recessive disorder subtype is characterized by severe bleeding in early childhood. In addition to mucocutaneous bleeding symptoms, found in the other subtypes, patients also experience frequent joint and soft tissue bleeds. Molecular genetic studies indicate the disease includes a variety of gene defects, including large deletions, frameshifts, and nonsensical mutations in the vWF gene.21

Shear Stress Fluid Mechanics Theory

Shear stress is the component of the stress tensor in the same plane of a materials’ cross section. Viscosity can be defined as the force a flowing fluid exerts on a body in the flow direction, providing resistance. In a Newtonian fluid, shear stress and viscosity have a direct relationship and in order to obtain the relation, a fluid layer between two very large parallel plates separated by 푙 is considered. A constant parallel force 퐹 is applied to the upper plate with a stationary lower plate, seen in Figure 5. The fluid velocity can be seen varying linearly from 0 to an arbitrary velocity V, exhibiting Couette flow. 9

The fluid in contact with the upper plate sticks and moves at the same speed, creating a shear stress 휏 acting on this fluid layer defined with Equation (1):

퐹 휏 = (1) 퐴 with A as the contact area of the region affected by shear.22 The fluid at the lower plate also assumes the plate velocity, zero in this case, due to the no-slip condition. The condition states that at the interface of solid and fluid, sticking occurs and causes the fluid at the face and solid surface velocity to be equal. The linear velocity profile and velocity gradient that is generated are shown in Equation (2).

푦 푑푢 푉 푢(푦) = 푉 푎푛푑 = (2) 푙 푑푦 푙 with y as the vertical distance from the lower plate. Within a time interval of 푑푡, the fluid particle sides rotate along MN through angle 푑훽 and upper plate moving distance 푑푎 = 푉푑푡, seen in

Figure 5. Angular displacement, or shear strain, is shown in Equation (3).22

푑푎 푉푑푡 푑푢 푑훽 ≈ tan 푑훽 = = = 푑푡 (3) 푙 푙 푑푦 10 Equation (3) can be rearranged to Equation (4), determining that the rate of deformation is equal to the velocity gradient.

푑훽 푑푢 = (4) 푑푡 푑푦

Thus, for a Newtonian fluid in one-dimensional shear flow, Equation (5) can be used.22 This indicates the rate of deformation is proportional to shear stress. 휇 is the constant of proportionality or dynamic viscosity (Ns/m2).

푑푢 휏 = 휇 (5) 푑푦

Shear stress can be obtained by multiplying the viscosity of the medium with the local shear rate.22, 5 For a Newtonian fluid, the shear force can be attained by combining equations (1) and

(5) to obtain equation (6).

푑푢 퐹 = 휏퐴 = 휇퐴 (6) 푑푦

While Newtonian fluids include water, air, gas and oil; non-Newtonian fluids include blood.

Thus, the relationship between shear stress and deformation rate is non-linear. Rather the slope

푑푢 of 휏 versus is the apparent viscosity of the fluid. Within the non-Newtonian realm, fluids can 푑푦 be further categorized by apparent viscosity or their trend of increasing or decreasing with the rate of deformation. For example, blood specifically carries characteristics of pseudoplastic and

Bingham plastic fluids. Pseudoplastic fluids are shear-thinning where the fluid becomes less viscous when it is subjected to higher shear levels. Bingham plastic fluids hold a yield stress, below which they are immune to shear, acting as a solid, and after which act as a fluid.22 Blood cannot be categorized into one category due to its complex and unique properties, but these classifications can serve as a guideline. 11 Ventricular Assist Devices

With an increasing number of implanted VADs, a deeper understanding of the shear and hemorheological effects need to be understood to improve the efficacy and safety of the devices.

Additionally, although long-term support is readily available, consistent clinical health risks need to be mitigated, particularly susceptibility to serious bleeding. This bleeding is a significant source of morbidity and mortality.23 While an anticoagulation regimen is prescribed post- operation, often an underlying non-treatable condition lies, symptomatic of AVWS.1 With potentially an unnoticed loss of large multimer vWF flowing in the blood, acquired Type 2 VWD could be overlooked and in need or clinical attention.

Dassanayaka et al. studied continuous flow and pulsatile flow VADs. The study suggests that although one of the mechanisms of vWFs degradation during VAD support is attributed to

ADAMTS-13, it is unlikely to be the only dominant degradation mechanism. From ADAMTS-

13 alone, the study showed a 5% increase in 225 kDa vWF monomers, while introduction of

VAD and shear flow had a 30% increase in the monomers. This suggests a separate and more robust mechanism responsible for vWF degradation. Potentially, that the introduced high shear stress may simply be tearing the disulfide bond between each vWF monomer apart. This indicates vWF has potential for other undiscovered degradation relationships. The study hypothesized future research is needed to elucidate the shear threshold after which platelet activation and vWF degradation occur in a VAD.4

In a retrospective study by Geisen et al., seven HeartMate II left ventricular assist devices

(LVAD) and five Thoratec biventricular assist devices (BiVAD) were compared against eight heart transplant patients. Regarding vWF levels post-procedure, large multimers were missing in the VAD patients while five of the six surviving heart transplant patients had normal multimer 12 patterns. Figure 6 shows the gel electrophoresis indicating vWF multimer varying for healthy versus VAD-implant patient.

Another complementary study by Meyer et al. studied the vWF profile and thromboembolic events in 51 patients with HeartMate II LVAD (HM II) and 51 patients with

HeartWare VAD (HVAD). These are the most frequently implanted VADs worldwide. The HM

II is an axial pump and the HVAD is a centrifugal pump. The study found that both populations had a trend of AVWS post-implantation. As a Type 2 VWD, it was observed that increasing

VAD operating speeds led to decreasing percentages of high-molecular weight vWF multimers, seen in Figure 7.24 This agrees with the characteristics of qualitative vWF defects found in Type

2 VWD. 13

Figure 7: Correlation between increasing VAD operating speed and decreasing percentage of high molecular weight vWF multimers; adapted from Meyer et al.24

With the amount of large, functional vWF decreased in VAD-implant individuals under high operating speeds, instances of epistaxis and gastrointestinal bleeding rose simultaneously.24

These are also indicators of AVWS and support the study’s correlation to AVWS.

Chapter 2

Optical Trap Theory

Optical traps are frequently used in biological applications. Through a highly focused laser beam, an attractive or repulsive force is administered dependent on the refractive index.

The piconewton force is able to hold an object in three dimensions within the focal point of the laser.25 The optical tweezers can manipulate micron-sized dielectric particles, like polystyrene beads, through the lasers small forces in its highly focused laser beam. The beam waist, seen in

Figure 8, contains the highest electric field gradient. This region draws dielectric particles to the center of the laser beam. However, a scattering force results in the particles’ displacement slightly downstream from the beam waist. This is due to the conservation of momentum, which states that the rate of change in the momentum of the light is equal and opposite to the rate of change in momentum, the force, of the object. Once the object is trapped in the Gaussian beam path; reflection, refraction, and scattering occur. Through balancing, scattering and gradient forces, the theory again harps on the conservation of linear momentum. Through these minute movements, it is evident that optical traps are very sensitive instruments, but provide an excellent tool while working on the biological micron level, like vWF or DNA. 15

In order to properly trap a bead, the laser forces applied on the bead must equal each other and, therefore, cancel out. This happens when the bead is centered within the trap and can be seen in Figure 9. The light gray arrows indicate equal forces and cancellation. They are pointed upwards due to Newton’s third law of equal and opposite reactions. The downward force, indicated by dark arrows, is attributed to the bead’s response to photon reflection, pushed with the momentum of the light proliferation. The arrow band through the bead can be credited to each photon meeting the refractive properties of the bead, similar to light passing through glass or water. 16

The optical trap theory measures light intensity to calculate the bead’s displacement. The theory assumes the displacement is a linear spring and is prescribed by Hooke’s Law, Equation

(7). 퐹 denotes the force applied by the laser, 푘 is the spring constant, and 푥 is the bead displacement.

퐹 = 푘푥 (7)

Chapter 3

Parallels between DNA and vWF

In order to confirm statistically and experimentally the future vWF optical trap study, a control study needs to be performed to test the optical trap theory and confirm the post- processing techniques. Double-stranded plasmid DNA was selected as a positive control due to plasmids affordability, relatively small size (compared to vWF), stiffness (discussed later), and simple linear structure. vWF is a complex and large glycoprotein, which undergoes an intricate sequence of post-translational alterations, including dimerization, glycosylation, sulfation, and multimerization.28, 29, 10 DNA provided a more reliable and scientifically understood molecule to conduct the study. Using DNA’s fixed structure that is less susceptible to cleavage or tear, DNA provided a simplified analogy to test the theories behind the vWF study. While the time constant of vWF unfurling and the speed of the conformational changes are unknown, the values are known for DNA.

In order to provide a range of shear effects on varying setups, three bead conditions were conducted: (1) DNA-coated beads of vWF length, (2) DNA-coated beads of smaller length, (3) non-coated beads. To determine the plasmids that correlate with these specifications, previous studies and published values were compared. The reference lengths were (1) 1.3 µm for filamental (unfurled) vWF, (2) 0.913 µm for a smaller plasmid.30, 5 Ideally, a plasmid of globular vWF-length (0.2-0.3 µm) would be used for the smaller plasmid, however plasmid’s of this length are not commercially available from New England Biolabs. 18 Effective Radius

The “effective radius” is used in this study to indicate the radius of the bead plus the

DNA strand lengths. In order to study the DNA under the optical trap, the effective radius extends from the center of the beads to the edge of the vWF/DNA attached to the bead. In Figure

10, the control study with DNA will resemble the “r3” bead because DNA does not have a coiled state, exhibited by ‘r2’. This is intended, as it provides a simplification for the control study.

Figure 10: Cartoon depiction of “effective radius” extending from the center of the bead to the edge of the DNA/vWF. DNA effective radii will resemble the ‘r3’ bead, adapted from Corsetti.31

The effective radius is based off of Hooke’s Law, Equation (7), involving the optical traps spring constant, and Stoke’s Law, Equation (8). Essentially, the force exerted by the trap on the bead (keeping the bead centered) is equal to the drag force exerted by the fluid flow on the bead surface. This means the trapping force equation equals the drag force equation (Stoke’s).

퐹 = 훽푣 (8)

The equation includes 훽 which equals 6흿흶r, the drag coefficient, where r is the bead radius, 흶 is the dynamic viscosity, and v is fluid velocity. After equating these two equations, the spring constant of the trap can be solved, shown in Equation (9).

푣훽 푣 ∗ 6휋ηr 푘 = = (9) 푥 푥 19 This calculation assumes the radius of the bead is 1 µm, prior to its measurement, a peculiar phenomenon discussed later. After the stiffness of the trap is calculated, the equation can be rearranged again to solve for the unknown effective radius. The effective radius is found through

Equation (10). The equation shows that a large displacement on the bead is related to a large drag force, and eventually provides a large effective radius.

푘푥 푟 = (10) 푣 ∗ 6휋휂

Objectives and Hypothesis

The objectives of this study are to (1) determine 2 plasmid-enzyme pairs with one of comparable length to unfurled vWF (1.3 µm) and one of shorter length (0.91 µm), (2) attach the treated enzymatically-cut plasmid to streptavidin polystyrene beads, (3) subject the beads to shear forces in the optical trap to compare effective radii to non-coated beads, and (4) determine the effect of shear rates on DNA-coated and non-coated bead effective radii. Thus, the study will serve as a positive control and protocol basis for a subsequent vWF study. The hypothesis is that there will be a quantifiable difference in bead effective radius between the DNA-coated beads and the non-coated beads. The bead radius is hypothesized to be independent of shear rates.

Chapter 4

Materials and Methods

The study is split into two phases: (1) preparing the DNA-coated beads and (2) subjecting the beads to shear on the optical trap. For phase 1, two double-stranded bacterial plasmids were attached to 2.0-2.9 µm streptavidin polystyrene beads (Spherotech Inc.; Lake Forest, IL) in separate solutions. Two bacterial plasmids, vWF length (1.3 µm) and shorter (0.9 µm), were selected, cut, and biotinylated to prepare for attaching to the streptavidin beads. In phase 2, the

DNA-coated beads were exposed to 2 shear levels (476.75 and 796.75 s-1) on the optical trap.

Shears were selected that do not eject the bead from the laser. Both DNA-coated beads were tested against control non-coated beads.

Preparing the DNA-Coated Beads

Selecting the Plasmid

To determine a plasmid of comparable length to vWF, each double-stranded plasmid base pair was determined to be approximately 0.34 nm.32 In order to match the length of ultra-large vWF catemer length of 1.3 µm, or filamental (unfurled) state, a plasmid with approximately

3823 base pairs was selected.30, 5 This is an estimate of vWF's length, due to homeostasis of the molecule making it extremely variable. The length was calculated through unit conversion starting from 1.3 µm and using the conversion factor of 1 base pair equaling 3.4 * 10-4 µm. 21 Plasmid pACYC177 (New England Biolabs; Ipswich, MA) was selected, which is 3941 base pairs or approximately 1.34µm, on par with the length of vWF.

Another plasmid, pUC19 (New England Biolabs; Ipswich, MA), of 0.913 µm length, or

2686 base pairs, was also selected. This is one of the smallest reproducible plasmids commercially available for reproduction and was selected as another comparison to the vWF plasmid-bead and the control bead.

Plasmid Digestion

While pUC19 is an isolated plasmid vector, the pACYC177 needed to be isolated via minipreparation. This isolates the plasmid from the E. coli K12 ER 2420. Once segregated into their respective plasmids, they were enzymatically digested to open into a strand. Restriction enzymes are proteins that can cut double-stranded DNA at specific palindromic sequence recognition sites. They cut the backbone of the plasmid recognition site, leaving an overhang. A multi-cloning site (MCS) enzyme was selected for each plasmid because it cuts at only one location, shown in Figure 11.

Multi-cloning site (MCS)

22 Restriction enzymes were selected with 3’ overhangs to mate with the subsequent 3’ biotin labels. MCS enzymes cut at one uniform site, thus opening the plasmid to a strand of equivalent lengths. DraIII enzyme (New England Biolabs; Ipswich, MA) met the qualifications for pACYC177 and pstI enzyme (New England Biolabs; Ipswich, MA) met the qualifications for pUC19.

To digest, 10X restriction enzyme buffer, restriction enzyme, and plasmid were thawed on ice. In a 50 µL tube, 5 µL of buffer and 4 µL of ddH2O (double-distilled H2O) were pipetted.

Eight-hundred units, approximately equaling 40 µL, of pUC19 were then combined. This ratio of plasmid to water is approximately the inverse of a standard plasmid digestion because this study required a large quantity of DNA. One µL, approximately 20 units, of restriction enzyme was added. The tube was then gently “flicked” and given a quick “touch” spin-down in a microcentrifuge. The tube was incubated at 37 ºC for 2 hours. Upon removal from the water bath, 10 µL of gel loading dye (New England Biolabs; Ipswich, MA) was added to stop the digestion reaction.

Purifying the Digested Product

Using a PCR (polymerase chain reaction) DNA Cleanup Kit (New England Biolabs;

Ipswich, MA), a 2:1 ratio of binding buffer to plasmid sample was combined and mixed by pipetting up and down. The sample was loaded into a column within a collection tube and spun in a centrifuge at 16,000 xg for 1 minute. Flow-through was discarded. Column was reinserted into a collection tube and 200 µL of DNA wash buffer was pipetted and spun for 1 minute. This step was repeated and flow-through was discarded. The column was transferred to a clean 1.5 mL microfuge tube. Six µL of ddH2O was added to the center of the matrix and set aside for 5 23 minutes and then spun for 1 minute to elute DNA. The cleanup kit protocol suggests using the kit’s DNA Elution Buffer, however, ddH2O was substituted in its place because it yields slightly higher volumes of purified digested DNA.

Biotinylation

Biotinylation is the process of covalently attaching biotin to a protein, nucleic acid, or molecule. In this study, biotin was attached to the 3’ ends of DNA plasmid, shown in Figure 12.

Figure 12: Cartoon depiction of cut plasmid with biotinylated 3’ ends

This process is rapid, specific, and mitigates the risk of perturbing the natural structure of the molecule, due to biotin’s minute size (molecular weight = 244.31 g/mol). Biotin has a high affinity for streptavidin, found on the streptavidin polystyrene beads, with a dissociation constant of 10-14 mol/L. The biotin-streptavidin relationship is the strongest non-covalent interactions in nature.33

A Biotin 3’ End DNA Labeling Kit (Thermo-Scientific; Rockford, IL) was used for the study. The kit uses terminal deoxynucleotidyl transferase (TdT) to incorporate 1-3 biotinylated ribonucleotides onto the 3’ end of DNA strands. While the labelling kit states the TdT exhibits a 24 substrate preference for single-stranded DNA and short DNA primers, it will also label duplex

DNA with 3’ overhangs, found in this study, albeit with lower efficiency. The kit also is optimized for labelling 1 µM of oligonucleotide (a polynucleotide whose molecules contain less than 200 base pairs), however, this study’s plasmids, >2,000 base pairs, were substituted.

To label the plasmids, kit components were thawed on ice. A portion of TdT stock was diluted from 15 U/µL to a working concentration of 1.5 U/µL by combining 2 µL of 5X TdT

Reaction Buffer, 7 µL of ultrapure water, and 1 µL of 15 U/µL TdT stock. In a sterile microcentrifuge tube, 25 µL ultrapure water (ddH2O), 10 µL 5X TdT reaction buffer, approximately 5 µL DNA (total yield from previous step), 5 µL Biotin-11-UTP, and 5 µL diluted

TdT (1.5 U/µL) were added. The reaction was incubated in a water bath at 37 ºC for 30 minutes.

Upon removal from the water bath, 2.5 µL of 0.2 M EDTA was added to stop the reaction. Fifty

µL of chloroform:isoamyl alcohol (24:1) was added to extract the TdT. The mixture was vortexed briefly and centrifuged for 2 minutes in a microcentrifuge to separate the phases. The top aqueous phase was removed and saved.

Attaching the DNA to the Bead

To determine the amount of beads to add to the DNA solution, the amount and concentration of DNA was calculated. First, two 2 µL samples of biotinylated DNA were tested on the NanoDrop 2000c Spectrophotometer (Thermo Scientific; Waltham, MA) to determine the mass concentration. The pACYC177 plasmid read 64.7 ng/µL and the pUC19 read 36.2 ng/µL.

By multiplying by approximately 41 µL of total DNA solution, the final DNA mass of the pACYC177 is 2652.7 ng and the pUC19 is 1484.2 ng. In order to convert this mass of double stranded DNA to moles of double stranded DNA 3’/5’ ends, several unit conversions were 25 required. NEBioCalculator, a New England Biolabs online molecular biology calculator, provided these calculations. Starting with Equation (11), the molecular weight (MW) of double- stranded DNA (dsDNA) was calculated.

푔 푀푊푑푠퐷푁퐴 = (# 표푓 푏푎푠푒 푝푎𝑖푟푠 ∗ 푀푊푎푣푔 푝푒푟 푏푎푠푒푝푎푖푟) + 36.04 (11) 푚표푙

The 36.04 g/mol accounts for the 2 –OH and 2 –H added to the ends. The average molecular weight of a base pair (푀푊푎푣푔 푝푒푟 푏푎푠푒푝푎푖푟) is 617.9 g/mol. The molecular weight and mass can then be applied in Equation (12) to calculate the number of moles.

푚푎푠푠 (푔) 푚표푙푒푠 = (12) 푚표푙푒푐푢푙푎푟 푤푒𝑖푔ℎ푡 (푔/푚표푙)

The number of linear dsDNA moles is multiplied by 2 to calculate the final number of moles of dsDNA ends. The pACYC177 was calculated as 2.179 pmol and the pUC19 as 1.788 pmol.

Assuming that all of the plasmid was bound to the beads at maximum binding capacity, addressed later in the discussion, calculations were made. The streptavidin polystyrene beads have a 0.5% weight/volume concentration and a binding capacity of 430 pmol Biotin-

Fluorescein (FITC) to 1 mg particle. Equation (13) was used to calculate the molarity.

0.5 푔 푠표푙푢푡푒 푏푒푎푑푠 430 푝푚표푙 1 푚푔 1 푚퐿 푝푚표푙 ∗ ∗ ∗ = 2.15 (13) 100 푚퐿 푠표푙푛 1 푚푔 푝푎푟푡𝑖푐푙푒 10−3푔 103휇퐿 휇퐿

With a molarity of 2.15 pmol/µL, Equation (14) was used to calculate the final volume of bead solution needed for each DNA solution.

1 휇퐿 푝퐴퐶푌퐶177: 2.179 푝푚표푙 ∗ = 1.013 휇퐿 푏푒푎푑 푠표푙푛 2.15 푝푚표푙 (14) 1 휇퐿 푝푈퐶19: 1.788 푝푚표푙 ∗ = 0.832 휇퐿 푏푒푎푑 푠표푙푛 2.15 푝푚표푙 26 The represented values in the equation were the values from the final solutions, from which data was collected. Prior to adding the beads, the beads were vortexed and sonicated for 30 minutes to achieve optimum particle suspension. Upon adding the final bead volumes, shown in equation

(14), the DNA-bead mixture was incubated in a water bath at 37 ºC for 15 minutes and then removed. Ideally, the mixture would be centrifuged/airfuged to separate the supernatant solution from the DNA-coated bead pellet at the bottom. However, the pellet was too small to be removed so the entire solution was kept. This was also in the attempt to promote further DNA binding to beads until testing.

A buffer, 1X phosphate-buffered saline (PBS), was added to increase the volume to 100

µL. Approximately 58 µL of 1X PBS was added to each DNA-coated bead solution. The two solutions were stored in the refrigerator until use in the optical trap, within one week. Prior to use, the solutions were sent through the pipette several times and flicked with a finger.

Running the Bead on the Optical Trap

Control Bead Solution Preparation

To make the solution of control non-coated beads to compare to the DNA-coated bead,

0.5 µL of beads were added to 199.5 µL 1X PBS. This solution was vortexed prior to use.

Flow Chamber Slide Preparation

A flow chamber holds a thin layer of bead solution to study on the optical trap. The flow chamber preparation was adapted from an established lab procedure from the Mechanobiology

Laboratory in the Biomedical Engineering Department at Penn State. On a Thermoscientific 27 glass slide, 2 strips of double-sided tape were spaced the approximate distance of a coverslip. A thin sliver of double-sided tape was placed through the center, as shown in Figure 13.

Left chamber Right chamber

Tape Figure 13: Flow chamber sealed on all four sides with the right chamber containing control beads and the left chamber containing DNA-coated beads. Two slides were constructed for the 2 DNA- coated bead groups.

A glass coverslip was placed on top of the tape, creating a square chamber with 2 open ends on each long-edged side of the slide. Excess tape was cut and removed using an X-ACTO knife. With a micropipettor, 15 µL of control solution was pipetted into the right side (control bead) chamber until the entire chamber surface area under the coverslip is covered with solution.

By ensuring no air bubbles are present in the chamber, incompressibility of the solution was assumed. After confirming that no solution has entered the other chamber (no visible water mark), the 3 sides of the right chamber are carefully sealed with quick-drying clear nail polish.

The nail polish is prudently applied, as to not touch any section of the left chamber. Fifteen µL of a DNA-coated bead solution was pipetted into left chamber and the remaining 3 sides are sealed. The slide is ready to be loaded onto the optical trap.

Optical Trap Setup 28 The slide is loaded onto the microscope and screwed in place with the coverslip side facing downwards. With each slide, the optical trap must be calibrated to determine the trap stiffness and strength. Within calibration, the bead is “trapped” by the laser, fixing its location in

3-dimensions. An external force is applied to the bead by the flow of the solution around the bead. No slip conditions are assumed, meaning the solution surrounding the bead is moving entirely in phase with the microflow chamber motion. Turbulence and backflow are assumed to not be present. The trap’s stiffness is related to the laser power, which creates a certain amount of drag force on the bead and moves the bead by a certain amount.34 If the trap has a lower power, or is less stiff, the bead will be able to displace a larger distance. The lateral bead movement in the direction of the fluid decreases the shear stress on the bead’s surface. Thus, the trap stiffness needs to be calculated in order to determine the amount of shear stress imposed on the bead. The displacement method was used to calculate the trap stiffness in the experiment.34

As stated earlier, the laser is modeled as a spring, according to Hooke’s Law in Equation (7). The trap stiffness is denoted by 푘, which is the spring constant of the trap. F is the force exerted on the trapped bead by the laser and 푥 is the bead displacement in one plane and direction.

The optical trap contains an IX71 Olympus epifluorescence inverted microscope with a computer-controlled piezoelectric stage, a Mad City Labs piezo tilt actuator, and a 635 nm picosecond pulsed laser, shown in Figure 14.35 A Cooke, Sensicam high speed, low light CCD camera captures the motion of the beads in the flow chamber.35 The camera was experimentally determined to have a resolution of 117.33nm/pixel.31 The microscope uses a 60x objective lens under the stage with oil immersion, calibrated to 23.17 ºC, the approximate room temperature.

The beads are imaged with the microscope set up for differential interference contrast (DIC). 29

QPD

Condenser

Stage

Objective (beneath Laser stage)

Camera

Figure 14: The optical trap consists of the IX71 Olympus inverted microscope with QPD, condenser, objective, stage, laser, and CCD camera.

The laser is controlled with the 6310 ComboSource Laser Diode controller set to 540 mA, shown in Figure 15. The laser is equipped with a 60 Watt TEC temperature controller.

Figure 15: Arroyo Instruments 6310 ComboSource Laser Diode controller set to 540 mAmps. 30

Trapping a Bead

A bead is trapped by moving the stage to have a single bead meet the laser location.

Through gently tapping the slide, the bead could be bumped into the control stream of the laser.

Once trapped, the bead is brought to the bottom of the slide, if it is not there already, by moving the objective knob. From the bottom, the bead is moved 15 µm up because the LabView programs are optimized to read effective radius at this plane. This is the location at which all testing will occur.

Quadrant Photo Diode Setup

With the slide loaded and microscope ready, the Quadrant Photo Diode (QPD) is calibrated. This ensures the alignment and accuracy of the data calculations. Incoming laser light passes through the bead, is recollimated by the condenser lens, and the QPD compares the output currents from all four quadrants to determine the positioning. Essentially, the QPD is responsible for position measurement through a relation between QPD voltage and bead displacement. The

QPD must be calibrated (centering the bead in the laser) for each bead prior to its testing.

The LabView program, “High Rate QPD Read and Setup D”, displays a live reading of the QPDx, QPDy, and QPDsum, shown in Figure 16. The QPDsum is maximized to 5.7, totaling the intensity signal being illuminated onto the QPD. While the QPDx and QPDy were previously assumed to need zeroing, it was determined through trial and error that zeroing the QPDx and

QPDy is less important than maximizing QPDsum. There was better independence between x and y positioning by maximizing the sum than zeroing the x and y. The 3 are all related so adjusting one QPD, adjusts the other values. Therefore, the experiments ensured the QPDsum 31 was maximized and the QPDx and QPDy were approximately 1.75. The QPD x and y values were held consistently for all experiments. Figure 16 shows the QPD x and y slightly separated, for reader clarity. The image is taken prior to overlaying them to the same value for the experiments.

Figure 16: QPD LabView program live preview with QPDsum (green line) maximized and QPDx and QPDy (red and white lines). QPD x and y are slightly separated to display the two lines, for clarity.

Spring Constant Calculation

To determine the stiffness of the trap, the spring constant is calculated. Five beads from each slide are trapped, QPD calibrated, and used to calculate at least five spring constants and conversion factors (in µm/V). Both values will be used in a later effective radii calculation.

These are then averaged and used for the data collection and calculation for that entire slide. The

LabView program “Multi-Function-Synch AI-AO-Trap_Curve_Fit_Sine_tone_edit” was used for spring constant calculation, shown in Figure 17. The program is set to sinusoidal oscillation 32 with an amplitude of 1 V. The same setup, sinusoidal oscillation at 1 V, is also used for the subsequent data collection.

Pre-Shear Rate Generation

Once the QPD and spring constant have been calibrated, the beads can be pre-sheared.

The LabView program “Triangle Waveform Trap Calibrator” is used. This involves subjecting the beads to specified amplitude and frequency using a triangular waveform of stage motion in order to reach a certain shear level. The shear level can be changed depending on the amplitude and frequency. This pre-shear will be important in future studies that will require vWF to reach a certain shear value, testing for unfurling. Although the shear wave is oscillating, only the maximum shear, or peak shear, will be used in calculations. 33 The stage velocity is used to calculate shear rate. Stage/fluid velocity for a triangular waveform is calculated by dividing the distance the fluid and stage travel by the time it takes to travel the distance. The amplitude correlates to the displacement and can be divided by one- fourth times the inverse of the frequency, the time.31 The one-fourth accounts for the amplitude being achieved in the first quarter of the waveform motion, shown in Figure 18. While the velocity is only calculated for the first quarter of the wave, the magnitude is constant for the entire waveform. The velocity merely oscillates from positive to negative.

Figure 18: Triangular waveform used in stage oscillation in pre-shear program, adapted from Corsetti.31

The amplitude voltage is multiplied by 10 to convert to microns because an increase in 1

V on the QPD readout equals a 10 µm displacement. The velocity equation can then be calculated in Equation (15).

휇푚 휇푚 퐴푚푝푙𝑖푡푢푑푒 ∗ 10 푣푒푙표푐𝑖푡푦 ( ) = 푉표푙푡 푚푎푥 1 1 (15) 푠 ∗ ( ) 4 푓푟푒푞푢푒푛푐푦

Using velocity, the shear rate was estimated by fitting a linear regression between calculated velocities and computational shear data, shown in Equation (16) and adapted from Corsetti.31

푠ℎ푒푎푟 푟푎푡푒 (푠−1) = (2 × 106 ∗ 푣) − 3.2516 (16) 34 The linear regression line in Equation (16) uses the maximum velocity (in m/s) calculated in

Equation (15) with a slope of 2×106 and a y-intercept of -3.2516.

Table 1 shows the experiment’s amplitude and frequency values and their corresponding fluid/stage velocities and shear rates.

Table 1: Input Amplitude and Frequency with the corresponding stage velocities and shear rates

Amplitude (V) 3 5

Frequency (Hz) 2 2

Stage Velocity 240 400

(µm/s)

Shear Rate (s-1) 476.75 796.75

There is a threshold at which the laser does not have enough force to hold the bead in the trap. At this point, the bead would be ejected from the trap. Due to the laser power being decreased to

540 mA (from previous studies 1000 mA), the laser weakened its ability to hold the bead in the trap.31, 26 However, this decrease in power improved the trap function. Past studies showed the trap was too stiff when set to 1000 mA. This was confirmed in screenshots of the LabView programs, which demonstrate the bead experiencing a static constant line rather than a sinusoidal motion with the stage.26 Since the laser was at such a high current/power in past studies, the bead was unable to effectively oscillate within the laser, producing a static constant line. This is more difficult to study since the minute motion of the bead in a stiff trap cannot successfully be modeled as sinusoidal. Through decreasing the laser power, the bead was able to achieve sinusoidal movement, but the compensation was the ability to achieve high shear rates.

Therefore, this study could not produce shear rates higher at 1253 s-1. 35 Figure 19 shows the LabView pre-shear program used. Each bead is pre-sheared 3 times for 10 seconds at one frequency and amplitude specification. All beads were pre-sheared in the x-direction but the figure demonstrates the pre-shear program can move the stage in either the x- or y-direction by selecting the green arrow (beneath the amplitude value). The independence between the QPDx and QPDy waveform was in direct response to maximizing the QPD earlier and leaving the QPDx and y as previously described.

At each shear level, 10 control beads and 10 coated beads were tested and subjected to data collection testing.

Post-Pre-Shear Data Collection 36 After pre-shearing the bead at the specified shear rate, data were collected to determine the effective radii of the beads. The same sinusoidal stage motion with amplitude of 1 V that was used in the spring constant program was used for data collection. LabView program “Multi-

Function-Synch AI-AO_Trap_Curve Fit_Sine_Tone_Phase Detection” was used and is shown in

Figure 20.

The stage motion oscillation creates a relatively small drag force on the bead, which displaces the bead slightly from the center of the trap. The QPD reads this displacement along with the digital images recorded by the CCD camera, shown in Figure 21. 37

The program outputs phase shift (degrees) between bead motion and stage velocity and the displacement of the bead (Volts). The phase has no known relation between shear conditions, as studied by X. Candela.26 While the phase data were saved for these experiments, it was not explicitly used for calculations and data analysis. The bead displacement is converted to µm using the µm/V conversion value obtained from the spring constant program. Data are collected for approximately 3 minutes as the stage oscillates for a total of 20 sinusoidal cycles. The bead motion is recorded over the cycles and the program outputs 10 values. After the bead is measured, the program prompts the user to trap another bead and repeat the process. Ten different beads were studied for each solution and shear rate.

The experiment consisted of 4 bead conditions: non-coated and DNA-coated beads at low shear (476.75 s-1) and high shear (796.75 s-1). The non-coated beads served as a control to determine if there is an effective radius difference between DNA-coated beads. The radii were hypothesized to be independent of shear rate.

Chapter 5

Results

Two slides were used to collect data. Both slides contained one flow chamber with non- coated control beads and the second flow chamber with one of the DNA-coated bead samples.

To calculate the effective radius, the spring constant and bead displacement were experimentally calculated and used.

Spring Constant

The spring constants and conversion factor (µm/V) were computed for both slides and applied only to its respective slide. Five non-coated beads on the pUC19 slide and 9 non-coated beads on the pACYC177 slide were trapped and their spring constant values were averaged. 9 beads were selected on the latter slide to determine the effect of increasing the sample size on decreasing the standard error of the mean. The averaged values and standard error of the mean can be found in Table 2.

Table 2: Spring Constants and Conversion Factors for both DNA-coated bead slides

Spring Constants Conversion Factor

(pN/µm) (µm/V)

Average 24.16 0.2781

Std Error of the pACYC177 Slide Mean 0.6136 0.0054 39 Average 25.11 0.3434

Std Error of the pUC19 Slide Mean 0.6995 0.0169

The table shows relatively similar spring constant values of 24.16 and 25.11 pN/µm for the two slides. The conversion factor is 0.2781 µm/V and 0.3434 µm/V for the pACYC177 slide and pUC19 slide, respectively. The differences between the two slides indicate the importance of calculating a spring constant and conversion factor for each slide. The values are dependent on the distance between the glass slide and coverslip and the bead size, which can vary when creating the microflow chamber slides.

Figure 22 shows a graphic representation of each bead trial and its corresponding spring constant. The pACYC177 slide (n=9) and pUC19 slide (n=5) are shown. The latter slide, indicated by orange points and best-fit line, had a smaller sample size and can be seen that its spring constant values decrease with each trial. This suggests that the spring constant value may be falling with each bead trial, or time (discussed later). Additionally, the data show that by increasing the sample size (n=9), for the pACYC177 slide, the line of best fit became more constant even though the distribution in values was larger. While both trend lines show a decrease in spring constant values, this could be attributed to the small sample size or, potentially, the changing power of the optical trap. 40 28

27 y = -0.9524x + 27.968 R² = 0.9269 26 pACYC177 Slide

25 y = -0.2197x + 25.263 pUC19 Slide R² = 0.1068 24 Linear (pACYC177

23 Slide) SpringConstant (pN/um) 22 Linear (pUC19 Slide)

21 0 1 2 3 4 5 6 7 8 9 Bead Trial

Figure 22: Each bead trial and its corresponding spring constant, per slide. Lines of best fit and R2 values are shown.

Figure 23 shows another depiction of the spring constant distribution with a box plot and dot plot for the two slides. While the pACYC177 slide had a larger sample size (n=9), the value distribution was larger than pUC19 (n=5). 41 a)

22 SpringConstant (pN/um) 21 Dotplot of pACYC177 Slide, pUC19 Slide 20 pACYC177 pUC19 DNA-coated Bead Slide

pACYC177 Slide pUC19 Slide 21.6 22.4 23.2 24.0 24.8 25.6 26.4 27.2 Spring Constants (pN/um)

Figure 23: Spring constant distributions for both slides shown in a) box plot and b) dot plot form. pACYC177 spring constants (n=9) and pUC19 spring constants (n=5)

Bead Displacement

The data were collected from two slides (one with pACYC177 and one with pUC19) with four bead conditions each. Each slide had data from non-coated at 476.75 s-1, non-coated at

796.75 s-1, DNA-coated beads at 476.75 s-1, and DNA-coated at 796.75 s-1. There were 10 beads

(n=10) studied for the groups except for the pUC19 slide populations DNA-coated beads at

476.75 s-1 and non-coated at 476.75 s-1, which had 11 beads (n=11). For each bead studied, 10 measurements (displacement and phase) were taken. All 10 measurements were averaged, which 42 was then used in the data analysis. Figure 24 shows the averaged displacement values, in Volts, from the center of the optical trap for both slides.

1.2

1.0

0.8

Control Bead 0.6

0.4 DNA-bead (vWF-length plasmid :

Displacement Displacement (V) pACYC177) 0.2

0.0 0 200 400 600 800 1000 Shear (sec^-1)

1.2

1.0

0.8

Control Bead 0.6

0.4 DNA-bead (shorter length

Displacement Displacement (V) plasmid : pUC19) 0.2

0.0 0 200 400 600 800 1000 Shear (sec^-1)

Figure 24: Displacement (V) values of the bead from the center of the trap. (A) pACYC177 slide (B) pUC19 slide. Standard error of the mean bars are shown.

43 The two plots show the 4 conditions for both of the slides. Standard error of the mean are shown. The non-coated beads showed lower displacement values from the center of the trap, as expected, with displacements of 0.9208 V, 0.8652 V, 0.7392 V, and 0.7976 V across the control groups on both slides. These values are all smaller than the displacements of the DNA-coated beads. The pUC19-coated bead had average displacement values of 0.8677 V for the low shear and 0.9258 V for the high shear. The pACYC177 plasmid had average displacement values of

0.9994 V and 1.0577 V, larger than the control and pUC19 plasmid, as expected. The bead displacement showed an increase with the bead’s effective radius. This can be attributed to the larger radii producing larger drag forces in response to the laser trapping the bead in place.

There is an increase in displacement with increasing shear rates, except for the control bead on the pUC19 slide. For the pACYC177 slide, the increase with shear rate is the same for both the non-coated and DNA-coated bead. The pUC19 slide shows an increase in displacement with shear for the DNA-coated beads and a decrease in displacement with shear for the non- coated beads.

Effective Radius Values

As previously described, the effective radius equation was derived by setting Hooke’s

Law and Stoke’s Law equal to each other. The spring constant and radius equations are provided again.

푣훽 푣 ∗ 6휋ηr 푘푥 푘 = = (9) 푟 = (10) 푥 푥 푣 ∗ 6휋휂

44 The k is the averaged spring constant for each slide (pN/µm), x is the displacement using the spring constant conversion factor from microns to Volts (µm), 휂 is the fluid dynamic viscosity PBS of approximately 10-3 Ns/m2, and v is the fluid/stage velocity (µm/s).36

Applying the spring constant, conversion factor, and displacement, the effective radius (r) can be calculated. Figure 25 shows the calculated effective radius values for the two slides with different bead conditions.

1.4

1.2

1.0 µm)

0.8 Control Bead 0.6 DNA-bead (vWF-length plasmid :

0.4 pACYC177) Effective Effective Radius( 0.2

0.0 0 200 400 600 800 1000 Shear (sec^-1)

45 b)

1.6

1.4

1.2 µm) 1.0

0.8 Control Bead

0.6 DNA-bead (shorter length

0.4 plasmid : pUC19) Effective Effective Radius(

0.2

0.0 0 200 400 600 800 1000 Shear (sec^-1)

Displacement is directly proportional to effective radius, so there is evident similarity between Figure 24 and Figure 25. Figure 25 illustrates the effective radii of the control beads on both slides at high and low shears are 1.045 µm, 0.9819 µm, 1.0763 µm, and 1.1612 µm. These values are roughly in the expected range of radii for control beads, 1.0 – 1.45 µm, as per the polystyrene bead manufacturer’s technical specifications. Within the range, the majority of the beads provided by the manufacturer have a radius of 1.065 µm, which is more consistent with the data shown.

Figure 25(a) shows the pACYC177-coated bead with an effective radius of 1.1341 µm at low shear and 1.2004 µm at high shear. The theoretical effective radius is 2.37 µm, which is not achieved. There are several explanations and theories highlighted later in the discussion.

However, while the effective radius does not reach the theoretical value, there is still an observed 46 increase in radius compared to the slide’s non-coated beads, 0.9819 µm and 1.045 µm. This implies that DNA has been coated onto the bead but the amount and distribution of the coating remains largely unknown.

Figure 25(b) shows the pUC19 DNA-coated bead with an effective radius of 1.2634 µm at low shear and 1.3479 µm at high shear. The theoretical effective radius is 1.98 µm, which is also not achieved. However, this DNA-coated bead also showed an increase in effective radius over its non-coated beads, 1.1612 µm and 1.0763 µm. The same implications and discussion can be repeated for this DNA-coated bead.

Chapter 6

Discussion

The data show (1) successful calculation of the effective radius of control beads and (2) relative increase in effective radius for DNA-coated beads compared to their respective control beads. Several theories and explanations can be made regarding the results inconsistency between the theoretical DNA-coated beads effective radius and the experimental radius.

DNA-Coated Beads Analysis

Amount of DNA Coated on the DNA-coated Beads

The exact DNA coating of the bead is largely unknown, at this point. While the coating has not been determined, the data show there is an increase in effective radius compared to non- coated DNA-coated beads. The effective radius may not be as large as the theoretical value for several reasons. Some suggestions include: (1) the DNA is sparsely distributed on the bead, (2) the DNA is clustered on only one side of the bead, (3) the DNA is bending (not rigid), and (4) the amount of DNA attached may vary on each bead. These factors are related to each other. For example, if DNA is sparsely distributed, the DNA could have room to bend or rotate, thus potentially lowering the effective radius value. If the DNA were densely packed, the DNA would be more susceptible to being straight due to the smaller space available for each DNA.

Additionally, this study assumed that each step in creating the DNA-coated beads was completed with full efficiency. However, it is unlikely that all of the plasmid were cut, all of the plasmid ends were biotinylated, and all DNA were attached to the bead. As noted earlier, the 48 biotinylation protocol exhibits a substrate preference for single-stranded DNA and only labels duplex DNA with lower efficiency. This all significantly lowers the amount of DNA that can be coated on the bead.

DNA Bending Rigidity

DNA was chosen for the purposes of this experiment because of its presumed high rigidity, one of the stiffest biopolymers, in comparison to vWF.37 While it is not fully understood as to why DNA has such unique rigidity, it is hypothesized that the electrostatic self-repulsion and compressive base pair stacking play a dominant role.37 By selecting a stiff material, the optical trap is expected to read the effective radius with more ease because the DNA-coated bead holds its shape.

However, one overarching concern to the study is that although DNA may be a stiff biopolymer, its actual persistence length value is smaller than expected. Considering that DNA is found in eukaryotes in extremely coiled states, DNA is capable of bending and wrapping. By selecting a circular piece of DNA, plasmid, it was presumed that its relatively small number of base pairs should be below DNA’s persistence length. Upon realizing the discrepancy in effective radius between the non-coated and DNA-coated beads, it was determined that double- stranded DNA has a persistence length of 50 nm, or 0.05 µm, which is below the length of plasmids used in this study.38 This could explain why there still was an effective radius increase between non-coated beads and DNA-coated beads, but it was not as large as the length of plasmids. DNA may have coiled-up or folded with the direction of the flow oscillating around it.

Since the length of the plasmid is larger than its persistence length, it is susceptible to folding. 49 LeDuc et al. data suggest that sheared DNA (as low as 0.2 s-1) can change the DNA orientation (within flow).39 Sheared DNA can orient itself perpendicular to flow (up to 90º), which is different from the expected ≤45º orientation, shown by other polymers. Applying

LeDuc’s data to this study, the DNA on the beads may be oriented in different directions (0-90º) with the flow. Since the DNA is not densely packed on the bead, this allows for unpredictable orientations and effective radii surrounding the bead.

DNA Under Shear

Shear forces have been found to have an adverse effect on DNA. Fortunately, studies suggest deterioration occurs at 1.4 * 10-5 s-1, which is several orders of magnitude higher than shear values used in this experiment.40 Nonetheless, DNA at low concentrations, as found in this study, are most susceptible to shear degradation.

Literature has also suggested that there are inconsistent experimental methods implemented between studies when researching DNA degradation under shear. This makes it difficult to find a definitive shear stress value that causes DNA damage.41 However, it is suggested that subjecting plasmids to shear stresses stemming from stirring, centrifugation, pumping, vial pilling, and filtration can be sufficient to degrade even highly-purified plasmids.41

Thus, the handling of DNA-coated bead solutions should be carefully intentional because it could have shear-induced degradation consequences.

Biotinylating Both Ends of DNA

The biotinylation procedure adds biotin to both ends of the digested plasmid. Due to the high affinity for streptavidin and biotin, there is the potential for both ends of the plasmid to 50 attach to the bead, forming a loop on the bead surface. This would cause the optical trap to read an incorrect effective radius by decreasing the plasmids length on the bead by half or less.

Additionally, the shear action of the optical trap stage could induce the other end of the DNA to bind to the bead, if it had not already done so. This would lead to a smaller effective radius.

Future studies could explore biotinylating just one end of the DNA by making the other end resistant to the biotin. Finzi et al. mentions a preparation that labelled one end with digoxigenin and the other with biotin.42 These are two of the most common labels for nucleic acids. By attaching the digoxigenin-labeled DNA ends to the glass coverslip, the study then attached avidin-polystyrene beads to the biotinylated ends. Another study, Zimmerman et al., used the location of a single adenine (A) in the overhang to tag the left and right end with biotin and digoxigenin, respectively.43 These studies indicate the potential for biotinylation of one end, rather than both ends.

Optical Trap Analysis

Variability in Manufacturer’s Bead Size

The manufacturer’s bead size variability ranged from 2.0 – 2.9 µm diameter. This variability could account for control beads and DNA-bead experimental effective radii. Figure 26 shows one CCD image example of the variability in bead sizes, with three different sizes pointed out by the arrows. 51

Figure 26: CCD image showing three different sized non-coated beads, demonstrating the variability in the manufacturer’s bead size

To combat this issue, beads were selected that were roughly the same size in the CCD camera image. However, future studies could more accurately measure the bead size with a camera scale bar or by determining the number of pixels in a bead. Additionally, 2.1 µm beads prior to DNA binding or beads with a smaller size range could be purchased.

Spring Constant Calculation

As mentioned, the spring constant is dependent on the distance between the slide and coverslip and bead size, which can vary over the slide surface area, and potentially with the operating time of the trap. This has been a preexisting limitation, which illustrates the importance of calculating different spring constant values for each slide. However, only 5 and 9 beads were selected as representative of both flow chambers of the slide, which may not be an illustrative sample size to determine the most accurate spring constant. Additionally, optical trap operating 52 time may be a factor in spring constant calculation, as it is suggested here that the spring constant value decreased with each bead, shown in Figure 22. The standard error of the mean for the spring constants on both slides was 0.6136 and 0.6995, shown in Table 2. The 95% confidence interval for the pACYC177 slide spring constant is ± 1.2027 pN/µm and ±1.371 pN/µm for the pUC19 slide. This means that there is a 95% chance that the sample mean will fall between

22.96 – 25.37 pN/µm and 23.74 – 26.48 pN/µm, respectively. Increasing the sample size for the spring constant calculation would decrease the range of potential spring constants and improve the accuracy of effective radius calculations.

Refractive Index

The optical trap uses refractive indices to discern the bead from the surrounding solution.

Therefore, the refractive index of the DNA and bead must differ from the surrounding PBS solution. According to the manufacturer, the polystyrene beads in this study have a refractive index of 1.59. DNA has a refractive index range of 1.5 - 1.75, which encompasses the bead’s refractive index value.44 The surrounding PBS solution has a refractive index value of 1.9, which is out of the beads’ and DNA-coated beads’ range.45 As long as the refractive index of the bead and DNA are different than the surrounding solutions’ refractive index, the laser and QPD will be able to distinguish the sample from the extraneous solution.

However, assuming there is non-cut or non-biotinylated or non-bound DNA floating in the solution around the DNA-coated beads, there is the potential for misperception by the system. There is the possibility that the optical trap may detect the refractive index of a nearby floating DNA and interpret it as part of the trapped beads effective radius. Using this assumption, there is the slight chance that this experiment’s DNA-coated bead data stems from 53 unsuccessfully coated beads (no DNA attached) and the optical trap detecting nearby DNA as part of the bead. However, due to this study’s consistent effective radii across the trialed DNA- coated bead, it is unlikely that this refractive index theory is of high importance or probability.

Premature Assumption of Bead Radius in Spring Constant Program

The LabView program assumes that the bead radius is fixed at 1 µm during spring constant calculation, which is before calculating the effective radius during data collection. This is a premature assumption because the radius cannot be set prior to the system calculating the radius. By assuming the bead will be 1 µm, the spring constant is computed and used for the effective radius calculation when, in fact, the effective radius may not be 1 µm. The threshold at which the difference between 1 µm and the actual calculated effective radius makes an impact is unknown and, thus, the effect of presetting this value is unknown.

Shear Rates and Displacement Calculations

For the pACYC177 slide, the increase in displacement with shear rate is the same for both the non-coated and DNA-coated bead. The pUC19 slide shows an increase in displacement with shear for the DNA-coated beads and a decrease in displacement with shear for the non- coated beads. The displacement and effective radius were hypothesized to be independent of shear rate. The radii discrepancy in non-coated beads at different shears may be caused by the larger drag force acting on the beads at higher shear rates. With a faster stage velocity at higher shear rates, the bead can be displaced further, resulting in a higher effective radius.

However, on the pACYC177 slide, both bead conditions experienced the increase in effective radius with shear. This suggests that the change in effective radius is not in response to 54 DNA length changing when shear is introduced. The DNA can be assumed to still remained intact without unfurling, breaking, or deteriorating. 55 Chapter 7

Future Work

This study is part of a larger study of vWF unfurling characteristics in response to shear stress. The present study’s data can be applied to the subsequent studies, as it has provided insight into the effective radii equation and provided a control on the optical trap.

DNA-Labelling Efficiency

There are several areas of this project that, if replicated, require adjustments. One adjustment is determining the labelling efficiency of DNA on the beads. Assuming that DNA can continue to be used for this project (i.e. neglecting its persistence length and propensity to bending), adding fluorophores to the 5’ terminal end of the DNA during the DNA-coated bead preparation would help determining labelling. This would assist in observing the bead under ultraviolet light in order to quantitatively determine the concentration of molecules present on each bead during post-processing. Additionally, fluorophores are used to mitigate DNA clumping and sticking. Another label that could be used to quantify that amount of DNA coated on the bead is YOYO-1, a green fluorescent dye used in DNA staining that can be observed under the microscope. However, it is speculated that it may adversely affect the entire bead’s drag coefficient, which would in turn affect the effective radius.

The labelling efficiency can also be checked during each step of the DNA-coated bead preparation. For example, gel electrophoresis can be used to determine and extract the amount of cut plasmid. The biotinylation procedure could also undergo its procedure for determining labelling efficiency. These steps would assist in removing the DNA that will not be able to bind from the solution. 56

An Alternative to DNA

While DNA-coated beads showed an effective radius change, ideally a stiffer polymer should be used to attach to the bead and determine the effective radius. Since the DNA length was longer than the persistence length in this study, the DNA was susceptible to bending. Future studies should select a polymer with a longer persistence length.

Optical Trap Studies

This study also introduced speculation about the non-linearity of the trap. Future studies should be conducted on non-coated control beads subjected to a range of shear values. These data will be used to determine if bead displacement changes linearly with changing shear rates.

This should be done prior to studying effective radii again to ensure that the fundamental operations behind the project are accurate. There may be a threshold at which the displacement and shear rates are no longer linearly related. This would also assist in better understanding the laser power to displacement relationship.

vWF Future Studies

An alternative to studying vWF on the optical trap would be placing the protein through a pump and using particle image velocimetry to study it. This pump could be simple network of pipes mimicking in vivo vessels with an LVAD. Through observing the protein trajectory, and potentially unfurling, through the flow, vWF can be analyzed quantitatively and qualitatively.

This would direct the future of this project towards previously conducted studies by Grant

Rowlands, studying vWF at the outlet of an LVAD.46 57 Finally, more data at higher shear rates would lead to improved replication of vWF unfurling in vivo conditions. On the optical trap, one way to combat this without vigorously oscillating the stage and potentially disturbing the alignment of the QPD and optical trap would be to jerk the stage in one swift motion while simultaneously imaging.26 Simultaneous imaging would be required because vWF can refold after shears are removed.

58 Appendix A

Displacement and Phase Raw Data - pACYC177

59 Appendix B

Displacement and Phase Raw Data – pUC19

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ACADEMIC VITA

Academic Vita of Maya A. Jankowska [email protected]

The Pennsylvania State University, University Park, PA; Graduating May 2017 Major: Biomedical Engineering; Minor: Engineering Mechanics Honors in Biomedical Engineering: A Control Study using DNA to Test the Effects of Shear Stress on Von Willebrand Factor Thesis Supervisor: Keefe B. Manning

Work Experience Abiomed (medical device company – heart pumps) May – Aug 2016 ACADEMIC RESEARCH INTERN Danvers, MA  Generated ~50 3D Mimics computer models from pediatric CT/MRI heart scans and studied correlation between demographics and heart dimensions in fit study  Developed laboratory protocol for mock circulation loop and troubleshot data acquisition for heart pump study  Initiated product failure investigation within research animal study

Hospital of the University of Pennsylvania May – Aug 2015 CLINICAL ENGINEERING INTERN Philadelphia, PA  Assisted device repair in-shop and on-the-floor in OR, ER, and nursing units in order to communicate, translate, and personally address clinicians product complaints  Trained to assist repair and preventative maintenance on Alaris infusion pumps and medical devices which resulted in decreased equipment downtime

U. Penn – Dept. of Pathology and Laboratory Medicine May – Aug 2014 ADNI BIOMARKER CORE LABORATORY INTERN Philadelphia, PA  Organized over 2,000 patient test tube samples that were analyzed to find biomarkers that could be early indicators of Alzheimer’s disease  Facilitated weekly 1-on-1 meetings with the department head to discuss new mass spectrometry journal articles and their potential application to the lab’s research

Grants Received Schreyer Summer Research Grant Summer 2014 Schreyer Study Abroad Grant Jan - Jul 2015  University of Freiburg, Germany; Schreyer exchange program Schreyer Honors College Academic Excellence Scholarship 2013 - Present

Leadership Experience Resident Assistant (RA: Atherton Hall) Jan 2016 – Present Leadership Assessment Center Aug 2015 – Present Schreyer Advancement Team May 2014 – Jan 2017