Force Image Cell Sensor Biomems Device Design Using PVDF Thin Film Thesis Presented in Partial Fulfillment of the Requirements F

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Force Image Cell Sensor BioMEMS Device Design Using PVDF Thin Film

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science

in the Graduate School of The Ohio State University

Edward A. Meier, B.S.

Graduate Program in Biomedical Engineering

The Ohio State University

2012

Thesis Committee

Professor Derek J. Hansford, Advisor

Professor Yi Zhao

Edward A. Meier

2012

Abstract

Researchers are predicting cancer will be the number one cause of deaths in the

United States by 2030. Therefore, better methods of detecting and diagnosing cancer cells are required. Further research has shown that cancer cells act very differently than normal cells, and that these characteristics can be used to detect and diagnose certain cancers. One such characteristic is the force exerted by cells undergoing tumorigenesis.

Polyvinylidene Fluoride (PVDF) has been of particular interest in recent years in bioMEMS devices because of its biocompatibility and piezoelectric properties. A device that takes advantage of PVDFs properties could measure these forces. Cellular force sensing can open new frontiers for diagnoses of diseases, better understanding of cellular mechanics, and a faster and less expensive than alternative methods of cellular force sensing.

This thesis presents the development of a sensor based on a microstructured piezoelectric thin film on a CCD to measure these cell forces. First, a model was designed to show the feasibility of such a device. The model provided a theoretical range of forces that can be expected from the device. The model showed that each pillar would be capable of measuring forces from about 26fN/s to 100pN/s, which correlated with the forces measure by other cellular force sensors. Finally, a prototype ii device was created as a proof of concept. This device reacted to force stimuli and gave results that were expected.

iii

Dedication

Dedicated to my mother, Debra.

Acknowledgments

I would like to thank Jeremiah Schley, Daniel Gallego-Perez, and Derek Hansford for their help in completing this project.

Vita

2003……………………………….. Greenville Senior High School

2008………………………………… B.S. Electrical Engineering Ohio Northern University

2008 to present………………. Master’s Degree in Biomedical Engineering

Fields of Study

Major Field: Biomedical Engineering

Table of Contents

Abstract ...... ii Dedication ...... iv Acknowledgments...... v Vita ...... vi List of Tables ...... viii List of Figures ...... ix Chapter 1: Introduction: ...... 1 Chapter 2: Piezoelectricity ...... 14 Chapter 3: Theoretical Model of PVDF Pillar Force Sensor ...... 23 Chapter 4: Experimental Methods ...... 34 Chapter 5: Experimental Results ...... 42 References ...... 50

vii

List of Tables

Table 1 Comparison of Force Sensors ...... 13

Table 2 Bond lengths and bond angles of β-phase PVDF42 ...... 20

Table 3 Ranges of the image sensor with an upper range at saturation of 100,000eV and lower range at the noise level of 15eV ...... 27

Table 4 Results from Model Design (a) Calculation of charge generated from single pillar; (b) Calculation of force sensitivity (slope of linear piezoelectric response); (c) Range of forces the sensor should be able to measure; (d) Range of forces per second for the sensor ...... 32

Table 5 Cost benefit analysis of various types of CCDs ...... 39

viii

List of Figures

Figure 1 Cellasic’s microfluidic channels are passively driven which eliminates the need for pumps and motors.7 ...... 3

Figure 2 Cellectricon High Quality Automated Patch Clamp: Left: wells are filled with buffer and compound, shown at left, and then pressurized with a lid; a patched cell is moved between the flows created by pressurizing the lid Right: shows the micro-channels from the wells to the patch-clamp8 ...... 4

Figure 3 Dynamic Array IFC showing microfluidic channels, chambers and valves and are reusable, decreasing costs9 ...... 5

Figure 4 IonFlux works by pushing cells through the main channel where they get trapped and introduced to up to 8 compounds. Then, electrodes (circled above) are measured using a patch clamp to produce the data on the particular ion channels.10 ...... 6

Figure 5 Sandia Labs silicon mirror and drive assemble (www.sandia .gov) .. 7

Figure 6 Sandia Labs MEMS device showing spider mite on gears (www.sandia.gov) ...... 7

Figure 7 Chemotherapy BioMEMS device that can control the release of the drug inside by heating up a coil16 ...... 9

Figure 8 Principle of magnetic twisting cytometry: the bead is magnetized in a known directions (Φ0), then the vertical magnetic field, H, twists the bead and the magnetometer records the change in magnetic field (B)35 ...... 10

Figure 9 From Harris, et al.: measurement of cell forces using a silicone rubber substrate being deformed by (a) single fibroblast cell and (b) cells spreading outward28 ...... 11

Figure 10 From Rajagopalan, et al. linear high-resolution bioMEMS force sensor: shows large deflections in probe without large deflections in the beams.26 ...... 12

Figure 11 From Ferrell, et al.: optical phase contrast image of polystyrene cantilever sensor at (a) 0min (b) 15min (c) 30min and (d) 45min shown prior to release of sacrificial layer. WS1 fibroblast cells are to be seeded into the middle of the device.27 ...... 12

Figure 12 Perovskite structure: smaller cations of titanium and zirconium are surrounded by the larger cations of lead39 ...... 15

Figure 13 Shows the effect of an electric field on a piezoelectric material (left) before applying the electric field V=n and (right) after applying the electric field V=n; shows an exaggerated effect of elongation (right) in the axis of poling on a material undergoing the poling procedure and the resulting orientation of the polar domains39 ...... 16

Figure 14 Matrices for piezoelectric materials: (a) elasticity matrix (b) dielectricity (c) piezoelectricity ...... 17

Figure 15 Crystal structure of α-phase PVDF41 ...... 19

Figure 16 Structure of β-phase PVDF (a) crystal structure showing distances between carbon chains (a and b); arrow indicates poling direction and (b) polymer chain diagram; arrow indicates poling direction42 ...... 20

Figure 17 Side by side comparison of α-phase and β-phase PVDF; Note the difference in polarity and symmetry between the two ...... 21

Figure 18 Unit cell model of force sensor showing the individual layers and the direction of the applied force which will be a 1nN test force: The top layers are composed of PVDF connected to the silicon base of the CCD with wire glue conductive adhesive ...... 24 x

Figure 19 Flat sheet of PVDF with force normal to the surface circuit diagram ...... 26

Figure 20 Wire Diagram showing the direction of the 1nN force in COMSOL; the lower arrows indicate that the unit is fixed on the bottom surface...... 28

Figure 21 Surface Charge Density in a Piezoelectric PVDF pillar surface charge density in C/m2 showing the inverse piezoelectric effect ...... 29

Figure 22 Bottom surface of PVDF pillar model (silicon layer) showing that that a surface charge is induced ...... 29

Figure 23 Voltage cross-sectional diagram of PVDF pillar model where the potential difference can be seen as a change of color from left to right ...... 30

Figure 24 The process of creating PVDF pillars from a silicon die. 1) the silicon die was created by photolithography 2) PDMS was added to the silicon die 3) the PDMS mold is allowed to cure for 48 hours and peeled from the die 4) PVDF is spin coated onto the mold 5) The PVDF pillars are removed from the PDMS mold ...... 35

Figure 25 Appearance of the conductive silicone from left to right: pressed from the tube, flattened with the lid of the petri dish, tapped into the counter lightly, spin coated at 1000 RPM, spin coated at 500 RPM ...... 36

Figure 26 Picture of the equipment for the poling process with the high voltage power supply (Left), double sided copper tape electrodes (right), and hot press (right)...... 38

Figure 27 DSP flow chart ...... 40

Figure 28 Picture from the original webcam (Logitech) before taking it apart ...... 43

Figure 29 Image from the webcam after removing the outer lens ...... 44

Figure 30 Image from the webcam with the PVDF thin film attached...... 44

Figure 31 Results from putting increasing pressure on the thin film using slightly increasing pressure from a pinky finger wearing a nitrile glove...... 45

Figure 32 Histogram of Figure 30 ...... 46

Figure 33 Histogram of Figure 31 (TOP) ...... 46

Figure 34 Histogram of Figure 31 (Bottom) ...... 47

Figure 35 Final Device showing lens housing removed, micropatterned PVDF thin film, and double sided copper tape: Logitech Quickcam ...... 47

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Chapter 1: Introduction

Purpose and Need

Currently, cancer is the second leading cause of death in the United States. The

CDC reports that there are 559, 888 deaths from cancer, second only to heart disease’s

631,636 deaths (2006). By 2030, however, deaths from cancer are projected to surpass those from cardiovascular diseases.1, 2 While our knowledge of cancer is growing at an increasing rate every year, there is much room for improvement. For instance, the methods for diagnosing cancer are still in their infancy. The tried and true method for diagnosing a particular cancer is still performed by a pathologist inspecting a slide of tissue that has been stained. This method is costly, time consuming, and rife with human error. Biomedical nanotechnology can provide faster, more reliable methods of cellular characterization and therefore the detection and treatment of cancer.

Therefore, doctors are looking to engineers for a solution to this problem.

Detecting cancer at the cellular level would greatly improve the ability of doctors to diagnose and treat cancer. Cancer cells have very different characteristics than normal cells.3 These characteristics have been quantified and can be used to make devices that differentiate normal cellular activity from those undergoing tumorigenesis.

One such cellular mechanism is the force generated by cells as they move and adhere to 1 surroundings. Research has shown that these forces are primary to cellular function.4,5

So, a device that makes use of a cancer cell’s unique force characteristics will be able to not only detect cancerous cells faster, more reliably, and less expensively, but expand our understanding of cellular mechanics.

MEMS for Cell Characterization

MEMS stands for microelectromechanical systems and encompasses a wide range of technological innovations, some of which we use every day. The accelerometers in automobiles and the ink jet printer are a few of the most recognized examples on the market today. It is hoped that MEMS to go further and revolutionize every facet of our lives, from faster networks using microscopic mirrors for fiber optic communications, to complicated bio-MEMS that can provide sensor data in the most sensitive parts of the body.6

Currently, there are no commercially available MEMS devices for cell characterization. However, there is a lot of research into cellular characterization, and a number of companies are able to provide different technologies that aid in the research and development of such devices. One company is Cellasic. They specialize in microfluidic technology for cell culturing, control systems, and screening. Cellasic’s process is borrowed from the semiconductor chip manufacturers, and relies on expensive “master templates,” inexpensive production of individual items at large quantities, and a consistent format.7 An example of Cellasic’s passive microfluidics is shown below in Figure 1. 2

Figure 1 Cellasic’s microfluidic channels are passively driven which eliminates the need for pumps and motors.7

Another company producing biomedical engineering solutions that help MEMS applications is Cellectricon. This Sweden-based company has paved the way for new

MEMS drug delivery systems in areas such as electrophysiology, microfluidics, microfabrication, and electroporation. The main target areas of research include: patch clamp screening, transfection, cell processing and analysis, and screening assays.

Cellectricon has combined these fields into two products, Dynaflow for electrophysiology, and Cellaxess for assays and transfection.8 An example of a Dynaflow

High Quality Automated Patch Clamp is shown in Figure 2.

Fluidigm is another such company that flourishes in MEMS research. Their focus, as their name suggests, is in fluid mechanics. Fluidigm’s co-founder, Stephen Quake, invented a microfluidic valve in 1998 at Caltech that was different from most other valves that relied on semiconductor manufacturing principles. Instead of using silicon, glass, or plastic substrates, Fluidigm fuses layers of rubber to make its microfluidic valves. Since this breakthrough, Fluidigm has produced a number of tools that would help in expanding MEMS research, such as TOPAZ, a protein crystallization package, integrated fluidic circuits (IFCs), Biomark, a microgenetic analysis system, dynamic array

IFCs Fluidigm 48 and 96, and NanoFlex valves.9 An example of a dynamic array IFC is show in Figure 3.

Figure 3 Dynamic Array IFC showing microfluidic channels, chambers and valves and are reusable, decreasing costs9

Fluxion Biosciences is another company that specializes in microfluidic research applications. Fluxion Biosciences has two main products, BioFlux and IonFlux. Bioflux, allows multiple reagents to be administered to cell arrays while providing a shear stress.

This can be used to support many field of MEMS and beyond, like cancer research, immunology, vascular biology, stem cells, and pulmonology. Ion Flux specifically allows the targeting of ion channel in cells. This is particularly useful in the drug development industry, especially those that target ion channels.10 A diagram of Ion Flux is shown in

Figure 4.

In 2000, one of the oldest labs in the country, Sandia National Laboratories started researching MEMS. Since then, the lab has produced some of the most breathtaking images of MEMS (Figures 5 and 6). More importantly, Sandia Labs has made strides in bioMEMS for cell characterization. Some notable advancements include: microfluidic diffusion coefficient measurement, microfluidic viscometers, filtration and separation membranes, cell manipulation devices, and microfluidic devices for electrophoresis and routing. 11, 12, 13, 14, 15

Figure 5 Sandia Labs silicon mirror and drive assemble (www.sandia .gov)

Figure 6 Sandia Labs MEMS device showing spider mite on gears (www.sandia.gov)

Even if there are commercially available bioMEMS, research into bioMEMS is ongoing. Some bioMEMS focuses on detecting and curing diseases. Researchers are finding new ways to incorporate bioMEMS into chemotherapy for cancer patients.16 An example of such a device is shown in Figure 7. Some researchers are looking at new ways to fight old diseases, like tuberculosis, with bioMEMS.17 It is no surprise that as diseases become more drug resistant, biomedical engineering will have to step up and replace old remedies. Researchers have even developed bioMEMS that may one day give sight to the blind.18

Researchers are not just interested in using MEMS for diseases, but for better tools to increase scientific knowledge. Some devices detect biomolecules in microfluidic channels that mimic body conditions.19 Also, researchers use bioMEMS to measure shear stress.20 Lab-on-a-chip designs provide faster, money saving results that are more mobile.21, 22, 23, 24 Other researchers have used BioMEMS for detecting biomarkers and pharmaceuticals.25 Force measurement is also of particular interest to scientists. Many researchers have been using bioMEMS to measure cellular forces.26, 27, 28, 29, 30, 31, These devices are discussed in greater detail later.

Figure 7 Chemotherapy BioMEMS device that can control the release of the drug inside by heating up a coil16

Cellular Forces

There are many different cells in the human body, all of which exert some kind of force on their surroundings to stay viable. Cells create forces by performing tasks responsible for their survival and function, like maintaining their shape, moving, transporting materials, and sticking to other cells or structures.32 Because these forces can be quite small and transient, cellular force measurement can be difficult.

There are a variety of ways researchers measure forces exerted by cells. One method is optical tweezers.33 Optical tweezers use a laser to provide the force which can then be measured by a microscope as deflections, usually using a tiny bead attached to the cell. Next, atomic force microscopes (AFM) allow force measurements of cells.34

AFM works like a record; a tiny needle attached to a cantilever moves over the surface of small objects, while a laser detects changes in the deflection of the cantilever. These deflections can then be translated into force measurements. Also, magnetic twisting cytometry (MTC) can be used to measure cellular forces.35 MTC uses magnetic beads to measure cell forces; the bead is magnetized in a known direction (Φ0), then the vertical magnetic field, H, twists the bead and the magnometer records the change in magnetic field as shown in Figure 8. Although they are useful techniques to measure cell forces, they are often costly, time consuming, only measure small forces and deformations

(optical tweezers, MTC, and AFM), and lower resolution (AFM with stiff cantilever).

BioMEMs offers a solution to these problems.

Many cell force sensors have been made using bioMEMs. Early work was done by Harris, et al., who used a substrate that was flexible enough to allow cell force interactions to be visible.28 This principle is shown in Figure 9. Another researcher,

Rajagopalan, et al., discovered that using a system of flexible beams, large forces could be detected by a probe without significant deflection of a probe.26 This point is illustrated in Figure 11. In another experiment, Ferrell, et al. used a polystyrene cantilever quad sensor array to measure single cell forces.27

Figure 9 From Harris, et al.: measurement of cell forces using a silicone rubber substrate being deformed by (a) single fibroblast cell and (b) cells spreading outward28

Figure 10 From Rajagopalan, et al. linear high-resolution bioMEMS force sensor: shows large deflections in probe without large deflections in the beams.26

Force Sensor Material Resolution Strength Range Fabrication Rajagopalan, et al. Si/Al 500pN,50pN 76Mpa 1µN Photolithography/ Cantilever ICP-DRIE Ferell, et al. Polystryrene 50nN n/a 1µN SLaM Beams Park, et al. SOI Cantilever 12nN n/a µN-mN DRIE Sun, et al. SOI Cantliever .01µN n/a Up 25µN DRIE Galbraith and PSG/Si .02µN n/a Various Micromachining/ Sheetz36 Cantilever photolithography Tan, et al.37 PDMS Micro 12 nN n/a 60nN Microcontact printing/ Needles photolithography Table 1 Comparison of Force Sensors

Summary

BioMEMS has taken a large leap in recent years. Companies have produced research tools and manufacturing techniques that have taken the strain off researchers to develop their own tools. However, at this time there is still no commercially available cellular force sensor.

There are a variety of methods for measuring cell forces. Table 1 shows a list of various cellular sensors that are capable of measuring cell forces. Although each sensor is contructed differently, they can all basically measure the forces that have been found in research. However, there is still no senor that can give real time results of cells moving along a surface. This leaves room for a device that is capable of measuring cell forces in real time.

Chapter 2: Piezoelectricity

Piezoelectric Effect

The piezoelectric effect is of great importance to our everyday lives. From the

Greek word for pressure, piezo, the piezoelectric effect happens when a material is deformed and gives off an electrical field, and vice versa, when a material is submitted to an electric field, the material deforms.38 Therefore, piezoelectric materials can be called smart materials, or materials that react to their environment. What started out as a scientific curiosity, has evolved into a variety of everyday applications. One of the first uses was the quartz crystal in time keeping devices. Piezoelectric devices have since expanded to radios, televisions, inkjet printers, TV remotes, ultrasound transducers, car sonar, smoke detectors, and much more. Now, scientists and engineers are starting to realize the full potential of piezoelectric materials.

The first piezoelectric materials were those that were naturally piezoelectric, such as quartz, tourmaline, and Rochelle salt (potassium sodium tartrate). Presently, there are a variety of materials that exhibit the piezo effect. The most common piezoelectric material is probably lead zirconate-titanate (PZT). PZT has been extensively studied because the material has high piezoelectric and dielectric coefficients in certain phases.39 PZT and other ceramic piezoelectrics, like Barium titanate, have the perovskite 14 structure. Shown in Figure 12, the perovkite structure has an oxygen octahedron with small cations of titanium, zirconium, tin, etc. in the octahedral A site (essentially Ti or Zr surrounded by six oxygen atoms), that is surrounded by the larger cations of lead, barium, strontium, calcium, etc. in the octahedral B site.

Figure 12 Perovskite structure: smaller cations of titanium and zirconium are surrounded by the larger cations of lead39

Poling

The structure of a crystal determines whether it will be piezoelectric. Crystal structure that lack a center of symmetry create poles that can then be aligned using an electric field. This lack of symmetry creates the means for positive and negatives ions to move in response to stress, which in turn gives the material its inherent properties of piezoelectricity. However, in some materials the individual crystals are oriented in such a way to cancel out the positive and negative effects. To fix this, researchers quickly

15 learned that the poles could be aligned when placed in a DC electric field. This process is known as poling. The process is illustrated in Figure 13.

Properties of Poled Piezoelectric Materials

The piezoelectric relationships are given by,

Di   ij E j  dijkTjk (2.1) for the piezoelectric effect, and

Sij  gijk Ek  sijklTkl (2.2) for the inverse piezoelectric effect where D is the electric displacement, T is the stress, S is the strain, s is the stiffness, and ε is the permittivity. Piezoelectric materials are initially isotropic, but after poling, the material becomes transversely isotropic in the poling direction. The matrices for elasticity, dielectricity, and piezoelectricity are show below. The variable of a transversely isotropic material are shown in Figure 14.

(a)

s11 s12 s13 0 0 0

s12 s11 s13 0 0 0

s13 s13 s33 0 0 0

0 0 0 s44 0 0

0 0 0 0 s44 0

0 0 0 0 0 2(s11-s12)

(b)

ε1 0 0

0 ε1 0

0 0 ε3

(c)

0 0 0 0 s15 0

0 0 0 s15 0 0

s31 s31 s33 0 0 0

Figure 14 Matrices for piezoelectric materials: (a) elasticity matrix (b) dielectricity (c) piezoelectricity

The most important parameters researchers use to gauge piezoelectric device effectiveness are the charge coefficients, d31 and d33, and voltage coefficients, g31 and g33. The relationship between these coefficients is given by,

d   *g mi nm ni (2.3)

Where m and n=1,2,3 and i=1,2…6. So, a high voltage coefficient, g, is useful in sensor applications while a high charge coefficient, d, is better for use in devices that require actuation, like vibration.

PVDF

Polyvinylidene fluoride (PVDF) has been investigated in recent years for uses in bioMEMs devices. Polymers like PVDF have gained popularity over the past few years for their biocompatibility, ease of production, and relatively low cost compared to silicon and ceramic materials.40 PVDF exists in different phases, α, β, ϒ,and δ, depending on certain conditions.41 Figure 15 shows the structure of α-phase PVDF. The β-phase is the most sought after for piezoelectric applications because β-phase PVDF exhibits the highest spontaneous polarization.42 Therefore, we will focus on β-phase PVDF. Table 2 shows the data on the structure and Figure 16 shows the structure of β-phase PVDF. A simpler view can be seen in Figure 17, which illustrates the difference between the two phases. The β-phase PVDF consists of a series of polar parallel chains while the α-phase

PVDF consists of non-polar “anti-parallel” chains.

Figure 15 Crystal structure of α-phase PVDF41

Bond Angle (Degrees) Bond Length (nm)

H-C-H 116.4161 C-C 0.15242

C-C-C 117.8 C-H 0.10789

F-C-F 109.5665 C-F 0.13457

Table 2 Bond lengths and bond angles of β-phase PVDF42

Figure 17 Side by side comparison of α-phase and β-phase PVDF; Note the difference in polarity and symmetry between the two43

Poling PVDF

PVDF has been poled in a variety of different ways. One way to pole PVDF is by embossing. Embossing works by pressing a material into a mold with enough pressure to induce poling by thermomechanical history and crystal nucleations.44 However, this method by itself only produces the δ and ϒ phases of PVDF.45, 46 Another method is electrical poling. This method produces the most β-phase PVDF and is the phase most sought after by researchers. However, a large DC electrical field is needed to pole the

PVDF completely and takes a long time, usually 3-5 hours. A more effective way to pole

PVDF is by increasing the temperature. Increasing the temperature of PVDF past the

21 melting point resets the structure of PVDF back to α-phase. The final way researchers have poled PVDF is by mechanical stretching.47 Mechanical stretching has been shown to be able to convert α into β PVDF for thin films. However, to maximize the effect of poling, most researcher choose to use a mixture of these polling methods, either combining heat and electricity, or pressure and electricity.

Chapter 3: Theoretical Model of PVDF Pillar Force Sensor

Introduction

The single cell model is the primary unit of the cell force sensor. Physics modeling software, like COMSOL, allows researchers to study piezoelectric materials.

This software is needed because each pixel of the CCD will be sensing just one PVDF pillar. While PVDF is not in the COMSOL library, the software allows user to create and customize materials. A simplified diagram of the device is shown below in Figure 18.

Using a unit cell model will also allow the sensor to be easily compared to other cell force sensors. This simple model will not only reduce computing time in COMSOL

(complex geometry/equations), but this unit cell model will allow us to look at a single

CCD pixel and come up with a force relationship on a pixel by pixel basis.

Charged Coupled Devices

Charged Coupled Devices, or CCDs, were developed at Bell Laboratories by

Willard Boyle and George E. Smith.48 In 2009, they received the Nobel prize in Physics

“for groundbreaking achievements concerning the transmission of light in fibers for optical communication."49 The device allowed the movement of charge from one capacitor to another. They were originally designed with memory systems in mind.

However, since the CCD sensors were sensitive to light, the most useful application has

23 been image sensors. Now, digital cameras using this technology can be found in almost every household nationwide.

Force PVDF Pillar

PVDF Thin Film

Copper Tape

Silicon/Silica

However, CCDs are prone to a noise from a variety of sources. The CCD has thermally generated electrons (dark current), bias voltage generated electrons (bias current), and optical effects such as dust. Dark current is usually corrected by taking an image with a closed shutter and subtracting it out or keeping the device extremely cool

(although in most cases this is not practical). Bias current, on the other hand, is corrected by reading the zero second exposure and subtracting that out.50 Optical effects are countered by keeping the system clean and free of debris. Although there are a variety of noise sources, the noise level is still very low, as we will see later.

Sensitivity

Sensitivity can be found by dividing the signal output voltage, VO, by the stress input, σ, placed on the PVDF,

V K  O (3.1) 

For a flat sheet of PVDF with a force normal to the surface, the sensitivity, is given by,

K1  g33 *h (3.2)

Where,

V g  O (3.3) 33  * h

where h is the height, or thickness, and g33 is the piezoelectric stress constant normal to the surface, of the film. However, a full electrical model of a pillar field is shown in

Appendix A.51 A simple diagram is shown below in Figure 19.

Figure 19 Flat sheet of PVDF with force normal to the surface circuit diagram51

Device Model

As discussed above, the bare CCD is very sensitive. Basically, each pixel is sensitive to even a few eVs (one eV is about 1.602*10^-19 Joules). That said, the model should still shows the ability to create a field several orders of magnitude about that. A per CCD pixel model of the device is shown below in Figure 20, showing the direction of the 1nN test force used. The model features a top layer of PVDF with one PVDF pillar, a layer of conductive adhesive (Cu in this case) and the silicon/silica layer of the CCD.

First, we need to determine the detection range for the CCD. The result of the model are shown in Figure 21. However, there is no accurate data on the CCD used in our application. So, the Kodak KAF-1603 1.6MP Full-Frame CCD image sensor will serve as a proxy.52 The noise level and the saturation level are given as 15eV (where eV is

26 equivalent to 1.602*10^-19 Coulomb*Volts) and 100,000eV, respectively. This range is shown in Table 3.

Charge of electron (C*V) Noise Level (C*V) Saturation (C*V)

1.602E-19 2.403E-18 1.602E-14

Table 3 Ranges of the image sensor with an upper range at saturation of 100,000eV and lower range at the noise level of 15eV

COMSOL multi-physics simulation software can be used to model the cell force sensor at each pixel. First, the model is sized to fit our device. The model is fitted with a

10 micron tall and 5 micron diameter pillars with a one micron this base. Then, the 1 micron thin conductive layer of copper is added. The final layer is a 1 micron thick layer of silicon. After that, the material needs to be specified. For this device, PVDF is used as the piezoelectric material. For our purposes, Xu et al. 2009 had all the information needed to create a model.

To measure the devices response, a test force of 1nN is applied to the model in the x-direction. Figure 20 shows a wire diagram of the direction of the force on the simulated PVDF pillar. This force can then be multiplied by a known force that has been seen in similar force sensor research, as discussed earlier.

Figure 20 Wire Diagram showing the direction of the 1nN force in COMSOL; the lower arrows indicate that the unit is fixed on the bottom surface

Results and Discussion

Figures 21 and 22 show the surface charge densities for the PVDF pillars. Figure

22 is used to illustrate that charge is found even on the grounded lower surface (CCD surface is used as a grounded 1 micron thick slice of silicon). The ranges that can be seen on the PVDF model are from 2.511*10-7 C/m2 to -3.498*10-7 C/m2, while the ranges that can be seen on the PZT-8 model are from 1.206*10-6 C/m2 to -6.666*10-7 C/m2. This is several orders of magnitude above what the CCD sensor can detect. Since the piezoelectric effect is linear, then you can simply multiply by the known cellular forces.

The Ferrell et al. 2011 polystyrene cantilever sensor has measured forces of 728 ± 74nN mean force per probe from skin fibroblast cells (ATCC) per 60 second time period. Also,

Figure 23 shows the voltage cross-sectional diagram where the potential difference can be seen as a change of color from left to right in the diagram.

Figure 21 Surface Charge Density in a Piezoelectric PVDF pillar surface charge density in C/m2 showing the inverse piezoelectric effect

Figure 22 Bottom surface of PVDF pillar model (silicon layer) showing that that a surface charge is induced

Figure 23 Voltage cross-sectional diagram of PVDF pillar model where the potential difference can be seen as a change of color from left to right

Next, from the surface charge density we can find the charge by dividing by the surface area. This can be seen in Table 4 (a). Now, since the piezoelectric response is linear, the line can be found by multiplying the Force (1nN test force) by the charge, found in Table 4 (b). From Equation 1, this slope also gives us the sensitivity of the sensor, at 39.8 *106 N/C. Then, the range of forces can be found by multiplying the force/charge of the sensor by the charge limit found earlier in Table 3. That gives the sensor a range of about 96pN to 638nN. Then, from Ferell, et al. we know that cells can generate from a few nNs up to approximately 600nN per probe per hour, or 167pN/s.

So, if the sensor read at 1/s to cut down on computer time analyzing the images, the 30 range of forces, with respect to time, would be 26.6fN/s to 177pN/s as shown in Table 4

(d).

(a)

Surface Charge Density (C/m2) Area (m2) Charge (Coulomb)

2.511E-07 1E-10 2.511E-17

(b)

Test Force (N) Charge from Model (C) Sensitivity (N/C)

1.00E-09 2.511E-17 3.98E+07

(c)

Force Sensor Range

Low End of Range (N) High End of Range (N)

9.57E-11 6.38E-07

(d)

Force Sensor Range with Respect to Time

Low End of Range (N/s) High End of Range (N/s)

2.66E-14 1.77E-10

Conclusion

This model shows that the image sensor should be capable of measuring the dynamic forces of cells. The range of forces cells exert of a half hour fall within the ranges of forces we can measure and with high sensitivity. Here, it has been shown that this force sensor design is able to measure cellular forces that have been measured by other force sensors. From this data, creating the device to measure cellular forces should be feasible.

Chapter 4: Experimental Methods Introduction

We have created a sensor capable of detecting cellular forces in the order of a few nanonewtons. This procedure for fabricating the device is shown in Figure 24. The process involves molding PDMS off a micropatterned silicon die, spin coating PVDF on to the mold, poling in an electric field, and applying the PVDF to the CCD.

First, a die was fabricated using the EV 620 Contact Aligner. The die was created from a silicon wafer and was first spin coated with SU-8 2005, a negative-tone, epoxy- type photoresist, at 1000RPM for 60s. The die was pre-baked for 4 minutes at 65°C.

Then the die was subjected to 7.5 seconds of UV through a micropatterned mask and the unexposed resist was washed away with SU-8 developer and isopropyl alcohol. After that, the silicon wafers were post-baked for 4 minutes at 100°C.

1) 2) 3) 4) 5)

Next, a PVDF solution was created by mixing PVDF powder (Aldrich) with dimethylacetamide overnight. Then, the mixture was stirred with acetone until the ratio of PVDF to solution is 10%. Next, the PDMS mold was created from the silicon die.

Conductive silicone (SSP779-NG(LV)) was to serve as the top electrode in poling the

PVDF. The mold was created by coating the die with the conductive silicone and allowed to cure for 48 hours. However, conductive silicone proved too brittle for use as a mold

(see Figure 25). To counteract this, the conductive silicone was mixed with 10:1 and 7:1 mixtures of PDMS to cross-linker in a 1:1, 1:2, and 1:3 ratio of PDMS to conductive

35 silicone. At these concentrations the PDMS was more moldable, but non-conductive. At the 1:3 ratio, the PDMS started to be brittle again, so conductive silicone was abandoned as a mold material. Finally, the plan was changed to use normal PDMS molded onto a silicon wafer that had been micropatterned. After a 48 hour cure, the mold was peeled from the micropatterned silicon wafer and served as a mold for the

PVDF.

Poling

The micropatterned PVDF film was pressed onto a glass slide and the removed and placed onto double sided copper tape. Next, the top electrode is created by placing another piece of double sided copper tape on a glass slide. The copper tape had to hang over the edge of the glass slide a little to allow the attachment of leads from the high voltage power supply. The copper tape was cut thin to reduce the chance of shorting.

Then, the two glass slides are brought together carefully to make sure that no copper is touching. A picture of the poling process is shown below in Figure 26. A high voltage power supply was then used to create a 100kV/m field for 3 hours at 160 degrees and 1 hour at room temperature. The micropatterned PVDF thin film then has to be carefully separated from the top electrode carefully with forceps.

Figure 26 Picture of the equipment for the poling process with the high voltage power supply (Left), double sided copper tape electrodes (right), and hot press (right).

CCD

The first thing that had to be done was to select the form factor for the CCD to be used in this device. CCDs have been used in a variety of devices and have also been made available wholesale. A table of the cost to benefit analysis is show below in Table

5. A webcam was chosen for its availability and low price. To make the CCD ready for use in this device, the outer casing and lenses were removed. However, the CCD had microlenses attached to it which had to be removed for signal transduction from the piezoelectric PVDF. To remove the microlenses, the cover-glass was removed and the

CCD was surrounded by PDMS. The PDMS served to protect the device from the piranha

38 etchant. Piranha etchant was created using a 1:1 ratio of sulfuric acid (Aldrich) and hydrogen peroxide (Aldrich). The piranha was carefully pipetted onto and carefully removed from the CCD until the microlenses were no longer seen under microscope

(approximately 1 hour). The CCD was then coated with a conductive glue (Wire Glue) to adhere it to the PVDF. The wire glue bonded to the poled PVDF, letting the poled PVDF slide out of the conductive silicone mold. However, in the final model, the microlenses were left intact to avoid damage to the CCD, and the wire glue was supplanted by copper tape.

Option Cost Pros Cons

Engineering grade CCD $1,500.00 Ease of Setup/Use Expensive

Cheap CCD Webcam $75.00 Low Cost, Ease of Use Some Setup, Low Cost

Building out own $500.00 Greater Configurability Costly, Time Consuming

Table 5 Cost benefit analysis of various types of CCDs

Digital Signal Processing (DSP):

Digital Signal Processing, or DSP, was used to make images more useable. Three particularly useful tools, are sharpening, denoising, and edge detection. For our purposes, the DSP is done in the webcam software. Edge detection was useful in finding the pattern that small cells make on the surface of the CCD. Denoising was useful to get rid of any graininess in the image. Finally, sharpening allowed the smallest signal to become very prominent. A flow chart of DSP is shown below in Figure 27.

Figure 27 DSP flow chart

Conclusion:

In conclusion, a device was created that will be used to measure cellular forces.

The CCD was selected based on its ability to convert the electrical impulses caused by deflection of PVDF pillars, to a readable signal, and in real time. However, a cost benefit analysis had to be done to determine which type of CCD would make the best sensor, with a webcam being the clear choice based on its availability, cost, and ease of operation. After selecting the right form factor for CCD, micropatterned PVDF was poled and applied to the CCD using copper tape. The device was now ready for testing.

Chapter 5: Experimental Results

Through the use of the piezoelectric properties of PVDF, we were able to show that a CCD camera could be used to measure the forces exerted by cells on top of the surface. Images were taken provided at every step of the disassembly and fabrication to verify that the camera was still functioning. These steps included: removing the lens, attaching the PVDF thin film with pillars, and applying forces to the thin film (on copper tape).

First, Figure 28 shows an image from the camera before it was taken apart. Next, we see in Figure 29 what the same image looks like after we removed the outer lens. To take apart the lens, one screw had to be removed to take apart the outer casing of the webcam. After that, the lens housing was removed by removing the two mounting screws located on the reverse side of the image sensor. Finally, Figure 29 shows what the image looked like with the PVDF thin film and copper tape attached to the surface.

To test the webcam to see if it could detect forces, a finger in a nitrile glove was used to lightly touch the surface. Then, the force was slightly increased. The results are shown in Figure 31. It appears as though the more force that is applied, the brighter the image becomes. To illustrate this point, Figures 32-34 show the histograms of figures 30 and 31 (top and bottom). Figure 35 shows the final device.

Figure 28 Picture from the original webcam (Logitech) before taking it apart

Figure 29 Image from the webcam after removing the outer lens

Figure 30 Image from the webcam with the PVDF thin film attached.

Figure 31 Results from putting increasing pressure on the thin film using slightly increasing pressure from a pinky finger wearing a nitrile glove.

Figure 32 Histogram of Figure 30

Figure 33 Histogram of Figure 31 (TOP)

Figure 34 Histogram of Figure 31 (Bottom)

Figure 35 Final Device showing lens housing removed, micropatterned PVDF thin film, and double sided copper tape: Logitech Quickcam

Discussion

The results showed that a device could be designed that responded to changes in force at the ranges epected to be exerted by cells. Images and histograms of the images taken reveal a change in brightness when a change in pressure is applied. For instance, the mean histogram of the first image, or the average pixel count at each bit (0 to 255), was only 1.3. After the first and second greater applied force, the means were

12.2 and 25.7, respectively. Also, the images from the webcam were blurred. This was most likely the result of using a thick layer of copper between the micropatterned PVDF and the CCD silicon, which caused diffusion of the piezoelectric response from the micropatterned PVDF pillars. The solution would be to use something that could be made thin, but still be conductive and tactile enough to remove the micropatterned

PVDF from the PDMS mold, such as wire glue (described earlier) or some kind of silver adhesive compound.

Conclusion

Material properties are very important in designing bioMEMS devices.

Polyvinylidene Fluoride has been of particular interest in recent years in bioMEMS devices because of its biocompatibility and piezoelectric properties. These properties have been harnessed to create a force sensor that is capable of sensing forces exerted by cells. Cellular force sensing can open new frontiers for diagnoses of diseases, better understanding of cellular mechanics, and a faster and more inexpensive than alternative methods of cellular force sensing. 48

Here, a model was designed to test whether such a device could be created. The model showed that this force sensor design was able to measure cellular forces that have been measured by other force sensors. Then, a device was developed that served as a proof on concept for the idea that a sensor based on a microstructured piezoelectric thin film on a CCD could be used to measure cellular forces. Therefore, it is plausible that future work will lead to a better developed device capable of measuring the forces of living cells on the PVDF surface.

Future Work:

The next step in the process will be to quantify the force exerted to produce the image seen in the camera. This can be done by a number of methods, like probe stations and atomic force microscopy. Then, the device can be used to measure the forces exerted by cells that move along the surface in real time. This would require water proofing the device to ensure that the circuitry is not damaged.

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