1. Cell Adhesion Study

8/12/2019 1. Cell Adhesion Study

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Final Project

ME 381 Fall 2006 Espinosa

Cell Adhesion Study Using MEMS and Digital Image Correlation

Keith Gall

Matt Schwabauer

Tareef Jafferi

Abstract:

Cell adhesion is an important area of research. Evaluating the mechanisms responsible

for cell adhesion could lead to dramatic developments in nearly all fields of medicine and

biology including cellular biology, drug delivery, and disease treatment. The following

paper describes new techniques formulated to study cell adhesion. Many current

techniques exist, but they have shortcomings. The proposed work addresses many of the

issues left unresolved by the existing methods. Furthermore, the proposed work will

reveal the mechanics responsible for cellular adhesion at a much greater resolution then

previously available. The following study builds upon existing technology by combining

the use of MEMS devices and Digital Image Correlation (DIC) in a creative new

approach. More specifically, the study will introduce a newly developed MEMS tensile

testing device that utilizes DIC to map 2-D strain fields within a cell, and adhesive layer

during applied static and oscillatory loading.

Introduction:

Advancements in MEMS (Microelectromechanical Systems), predominantly in

the past decade, have led to dramatic improvements in our everyday lives. Miniature

accelerometers detect crashes and activate airbags in our cars, miniature gyroscopes have


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improved the accuracy and reliability of control systems, and pressure sensors alert

drivers when their tires are getting low. The everyday practicality of these inventions is

obvious, yet these designs barely scrape the surface of possibilities for MEMS devices.

One of the most important and rapidly advancing areas of MEMS research is in

the field of medicine. Every day, with the help of MEMS devices, Scientists are

unlocking more secrets of the building blocks of life, cells. Just one of many important

properties of cells is adhesion. This is the attraction of a cell to any surface: similar or

dissimilar cells, proteins, fats, etc. Cell Adhesion can best be tested similar to the tensile

strength of other materials. Better yet, a dynamic testing machine can allow cyclic

loading, independent force of deformation, and adaptability for testing of various cells

connected to the device via any number of surfaces.

Many current testing devices monitor the critical parameters. Some studies even

use digital image correlation to monitor the deformation of microfabricated, support

pillars as the cell attempts to crawl across a surface. Other studies utilize a flat substrate

embedded with fluorescent nano-particles, then uses DIC to monitor the substrate

deformation as the cell attempts to crawl. However, in the proposed study a MEMS

device with two flat platforms being pulled apart by comb drives serves to introduce a

deformation and apply load to the bonded cell. By introducing the force, rather than just

observing cell crawl, this study can investigate a variety of parameters previously

unavailable. The device can pull an adhesively bonded cell in tension until failure to

reveal fracture properties. Additionally, the comb drives can apply oscillatory loading to

simulate real life conditions of a cell. Last, to quantify the deformation fields within the

cell, fluorescent nano-particles will be embedded in a substrate on the surface of the


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paddle-like tensile plates. This will allow DIC techniques to calculate 2-D strain fields as

the cell is being pulled in tension. Fluorescent nano-particles will also be embedded in

the cell itself to monitor deformation of the cellular structure.

Concept:

Our device will resemble a large-scale mechanical tensile testing machine in operation.

The forces will be applied unilaterally by two comb drives, one on each side. The

cantilevered test rods will each be supported by two bending beams, which will provide a

greater resistive force against the drive force. The cell will be loaded across two plates

coated in a deformable substrate and functionalized surface to promote adhesion. The

type of functionalization could vary depending on the type of cell being analyzed.


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Upon actuation of the comb drives, the plates will pull apart causing the cell to

deform. The clinging force of the cell to the protein will cause a shear deformation

within the substrate. The deformation within the cell will reflect both a normal and shear

stress.

The 2D deformations will be tracked using Digital Image Correlation.

Nanoparticles will be imbedded in the substrate in a random pattern and a camera will

monitor their movement. The cell itself will be stained in a random pattern so that the

membrane evolution can be monitored as well.

The device allows for various testing methods. The device can simply pull the

cell to failure, by applying a controlled voltage in the comb drive. Also, the device can

create cyclic loading conditions, in which the dynamic response over time is observed.


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This will provide very valuable insight into cell membrane properties previously

untested.

Digital Image Correlation

The method commonly referred to as Digital Image Correlation can has proven to

be a reliable, and flexible, tool for making displacement measurements. In general, the

DIC method tracks the location of points from a reference image and compares them to

the location of those same points in a new deformed image. To understand the DIC

technique, one must realize that a grayscale digital image is simply a 2-D array of values.

Each pixel represents a location in 2-D space and the value represents brightness or

intensity. However, since a single pixel location and value is not unique, DIC must track

a small subset of pixel values. At the core of the DIC technique then is a correlation

function that grades candidate subsets and displacements based on how closely it matches

the original subset. The most common correlation function is the Sum of Squared

Differences. Furthermore, since in real experiments displacements are not always 1 full

pixel, interpolation schemes are utilized. Cubic Polynomial, Cubic B-spline, and Quintic

B-spline interpolation functions are frequently used with higher order methods being

slower but generally more accurate.

C(x ,y , u,v ) = (I(x + i,y + j ) I* (i, j= n / 2

n / 2

x + u + i,y + v + j )) 2Pixel coord., reference image

Correlation

function

Displacement

n: subset size(5x5)

Image before motionImage aftermotion

Pixel value at (x+u+i; y+v+j)Pixel value at (x+i; y+j)

Sum of Squared Differences Correlation Function


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In this proposed series of experiments DIC will be utilized to track the location of

landmarks on within cell substructure. By treating the cell with certain chemicals,

particular areas of interest within a cell can be made to fluoresce. The type of chemical

staining depends on the type of cell, but has been successfully used in many recent cell

studies. In addition to using DIC to track cell deformation, this study will use fluorescent

beads embedded in a deformable substrate to track the cells adhesive response to

externally applied forces. The cell will be placed on two platens each composed of a

surface layer of polyacrylimide. This substrate will contain fluorescent beads that can be

tracked using DIC techniques. In this manner, the response of both the adhesive

properties of the cell and the cell substructure can be monitored during testing. By using

the fluorescent markers that emit a different wavelength of light, the DIC tracking

method can differentiate adhesive response of the substrate from the internal response of

the deforming stained cell.

Device Design Considerations:

An important design constraint is to use a comb drive to apply force on the

system. The comb drive allows for rapid (or slow) cyclic loading with ease. We were

initially concerned that it would not provide a large enough displacement to deform the

cell, however, this is not the case. This was our only major concern and was a reason we

considered the nanotractor, however the nanotractor is not as effective in cyclic loading.

It was determined that the maximum force of our device is 1.7 microNewtons

which is enough to separate a cell connected at around 40 points. This was determined

from the sum of the forces


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And then breaking down the comb and beam forces

This was all determined assuming that were using a structure made of Silicon.

This determines the stiffness, and is a variable we could change to fit different design

parameters. We could also modify the size or number of comb drives to create a larger

pulling force; the number of bent beams can also vary. All these features make the

design adaptable for a variety of conditions.

This design allows for a variety of tests to be run. We can run both static and

dynamic tests. Running an static could allow us to get a 3d perspective as it gives us time

to take capture images through the z-axis. In a dynamic test we can vary the frequency,

force, etc. All of these tests can either be stress controlled or strain controlled.

Like all sensitive testing equipment, our device should be calibrated. This can be

done using soft polymers or gels that will behave mechanically similar to cells. This will

allow us an easy way to run tests and generate a consistent baseline behavior. Because

our device is powered by a comb drive, we can apply a negative voltage to press the

plates together. This would allow the cell to be securely mounted on the substrate and

prevent the cell from crawling down the sides of the test platens.

Freaction = Fcomb Fbeam

F1comb =V2

2

w

z

= 3.53x10

9N Fbeam = Ktotal gap


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(e)

(f)

(g)

(a

(b)

(c)

(d)

Figure 1. Cross sectional diagrams

of comb drive fabrication

Fabrication

The fabrication for the cell adhesion study device is

based on fundamental techniques used in general

MEMs fabrication. For the simplification of describing

the fabrication process, the process has been divided

into two distinct parts: the construction of the comb

drives and the construction of the cell platform and

support beams.

Fabrication of the Comb-drives

To begin, copper is deposited on the glass substrate by

E-beam evaporation method (Fig. 1.a). A photoresist

(PR) is the coated on the copper layer and is patterned

as the etching mask of the copper (Fig. 2). The pole

layer of copper is patterned using wet etching

techniques in FeCl3etching solutions (Fig. 1.b). After

completing this step, Polyimide (PI) is spun as a

sacrificial layer and an anchor is defined. The PI is

cured at 150C to endure the next processing steps (Fig

1.c) (Mask for patterning displayed in Fig. 2). A seed

layer of Ti/Cu (300 angstroms/ 500 angstroms) for

electroplating Ni is deposited on the sacrificial layer (Fig 1.d). After depositing the seed

layer, a thick photoresist is spun and the comb is defined (Fig. 1.e). Ni is then deposited

for the finger layer by electroplating (Fig. 1.f). After this process, the seed layer of Ti/Cu


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Figure 2. (left) Mask 1, (right) Mask 2

(300 angstroms/500 angstroms) is etched by wet etching techniques in thin nitric acid and

Ti etching solution after stripping the photoresist with the use of acetone. The final

release step is performed by O2plasma dry etching

(Fig 1.g).

Fabrication of the Platform and Beams

The second part of our device, following the

comb-drives previously discussed, is the

platform in which the cell would sit as well as

the structural beams supporting the MEMs

system. Ni deposition and patterning is

conducted using Mask 3 (Fig. 4). After this

process, a Polyimide (PI) sacrificial layer is

coated and patterned. A layer of photoresist is

then spun, patterned using Mask 4 (Fig. 4), and

Ti/Au e-beam deposition and lift off is

Figure 3. Cross sectional diagrams of

cell platform and beam fabrication

(a)

(b)

(c)

(d)


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Figure 4. (left) Mask 3, (right) Mask 4

Figure 5. Mask 5 for the use of spin

coating a polymer on cell platform

conducted. After completion of this metal deposition, the sacrificial layer is removed

using O2plasma dry etching.

After the fabrication of the comb-drive as well as the cell platform and supporting beams,

a layer of polymer (varying depending on type of cell) can be spin coated using Mask 5

(Fig. 5) onto the platform. After the addition of the polymer to the cell platform, the

MEMs device is ready for cell placement and

adhesion tests.

Conclusions

By utilizing Digital Image Correlation

techniques this study aims to gather accurate data

on the mechanical response of cell structures. The


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design of a comb-driven MEMS test device will allow for a variety of experimental

parameters to be investigated. The simple design is based on many existing technologies

(comb drives, DIC) but combines them in a creative way that will allow for flexibility of

experimental variables. Overall, cell studies like the one proposed here, will prove to

contribute greatly to the understanding of many biological processes.

Documents

1. Cell Adhesion Study