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Microfluidics and Nanotechnology Final Report for
Fundamentals of Engineering Honors
Engineering 1282.02H
Spring, 2014
April 21, 2014
Team X6:
Turner Adornetto, Seat 32
Usoshi Chatterjee, Seat 29
Luke Forshey, Seat 30
Ryan Schoell, Seat 31
P. Clingan Monday 10:20PM
Date of Submission: 4/21/14
Executive Summary
The project described in this report includes two areas of focus: microfluidics and
nanotechnology. The microfluidics portion explored how yeast cell adhesion is affected by
changes in surface topography, specifically, patterned and smooth surfaces. Alternatively, the
nanotechnology portion was focused on the development of a theoretical NANOLYSER device
capable of accurately diagnosing a patient with a single blood sample collected using a
microneedle.
The microfluidics testing was performed using pressure driven flow. The yeast was incubated in
the chip channels and allowed to adhere to the channel. The yeast was then sheared off the walls
and chip channels using channel flow created through the Tygon tips and tubing. Five tests were
run using each chip base (the patterned and smooth) for a total of ten data points.
The NANOLYSER was created using research performed during initial brainstorming. The team
researched many different current medical nanotechnologies and used some of these ideas to
create a device to detect carriers as well as people who are infected with cystic fibrosis. The best
methods involved using electroosmotic flow to move the sample, a chemical lysis, pillars as
filters, protruding wedges to mix the sample, two heating and one cooling chamber to perform
PCR (Polymerase Chain Reaction), and gel electrophoresis and spectrophotometry to detect the
disease.
The microfluidics portion of this experiment resulted in an understanding that a patterned surface
with surface topology similar to that used in this testing would require about the same amount of
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shear stress to dislodge adhered yeast cells. The smooth chip base was used to standardize yeast
adhesion properties, while the pattern chip base with hexagonal wells was used as the variable.
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Acknowledgements
Team X6 would like to thank Mr. Paul Clingan, Andrew Theiss, Aaron Strickland, Martin
Spang, Erica Brackman, Ani Tarimala, and the rest of the FEH instructional staff. Team X6
would also like to thank the FEH program at The Ohio State University, and its sponsors for
providing this research opportunity.
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Table of Content1 Introduction..............................................................................................................................2
1.1 Problem Statement............................................................................................................2
1.2 Team Introduction.............................................................................................................2
1.3 Report Organization..........................................................................................................2
2 Microfluidics Background and Preliminary Concepts............................................................2
2.1 Background.......................................................................................................................2
2.2 Preliminary Concepts........................................................................................................2
2.3 Requirements and Constraints of Project..........................................................................2
3 SolidWorks Simulation and Fluid Flow Program to Develop Microfluidics Design Strategy2
3.1 SolidWorks Simulation.....................................................................................................2
3.2 Fluid Flow Program..........................................................................................................2
3.3 Flow Dependence on Applied Pressure............................................................................2
3.3.1 Results Obtained........................................................................................................2
3.3.2 Discussion..................................................................................................................2
3.3.3 Summary and Conclusions........................................................................................2
3.4 Decision Making Process..................................................................................................2
4 Microfluidics Experimental Design Brainstorming and Preliminary Testing.........................2
4.1 Brainstorming....................................................................................................................2
4.1.1 Chip Assembly...........................................................................................................2
4.1.2 Chip Design Changes................................................................................................2
4.1.3 Microfluidics Final Design........................................................................................2
4.2 Microfluidics Experimental Design..................................................................................2
4.2.1 Hypothesis.................................................................................................................2
4.2.2 Experimental Procedure.............................................................................................2
5 Microfluidics Results and Analysis.........................................................................................2
5.1 Results Obtained...............................................................................................................2
5.2 Discussion.........................................................................................................................2
5.3 Summary and Conclusions................................................................................................2
5.4 Calculations and Mathematical Analysis..........................................................................2
5.4.1 Results for Flow Rate and Shear Stress for Each Base Type....................................2
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5.4.2 Results in Terms of Hypothesis.................................................................................2
5.5 Sources of Error................................................................................................................2
5.5.1 Error within the Experiment......................................................................................2
5.5.2 Experimental Error....................................................................................................2
6 Microfluidics Summary and Conclusions...............................................................................2
6.1 Summary...........................................................................................................................2
6.2 Roadmap for Future Work................................................................................................2
7 NANOLYSER Design Background and Medical Application...............................................2
7.1 NANOLYSER Background and Chosen Medical Application........................................2
7.2 Brainstorming....................................................................................................................2
7.3 Device Structure................................................................................................................2
7.3.1 Testing Designs.........................................................................................................2
7.4 NANOLYSER Program....................................................................................................2
7.5 Summary of Relevant Work and Research Topics in the Field........................................2
8 NANOLYSER Strategy Development, Brainstorming, and Preliminary Design...................2
8.1 NANOLYSER Strategy Development..............................................................................2
8.2 Design Parameters.............................................................................................................2
8.2.1 Designs Considerations and Philosophy....................................................................2
8.2.2 Fabrication Considerations........................................................................................2
8.2.3 Biological and Biochemical Considerations..............................................................2
9 NANOLYSER Final Design....................................................................................................2
9.1 Explanation of Final Design.............................................................................................2
9.1.1 How It Works............................................................................................................2
9.1.2 Features......................................................................................................................2
9.2 Advantages of Design.......................................................................................................2
9.3 Disadvantages of Design...................................................................................................2
10 NANOLYSER Summary and Conclusions.............................................................................2
10.1 Summaries.....................................................................................................................2
10.1.1 Design........................................................................................................................2
10.1.2 Practicality of Chip Design........................................................................................2
10.2 Conclusions...................................................................................................................2
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10.2.1 Results in Terms of Purpose......................................................................................2
10.2.2 Roadmap for Future Work.........................................................................................2
11 Bibliography............................................................................................................................2
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List of Figures
Figure 1 Isometric View of the Channel Mesh Used In Flow Simulation.
Figure 2 Goals Plot Indicating Convergence of Maximum and Average Velocity for a
Pressure Difference of 49,000 dyne/cm2.
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Figure 3 Cross Sectional Velocity Contour for a Pressure Difference of 49,000 dyne/cm2.
Figure 4 Shear Stress Contour for a Pressure Difference of 49,000 dyne/cm2.
Figure 5 Microfluidics Program
Figure 6 Graph of Theoretical Flow Rate versus Water Height.
Figure 7 Graph of Theoretical Pressure versus Water Height.
Figure 8 Graph of Theoretical Flow Rate versus Theoretical Pressure.
Figure 9 Chip and Chip Holder Assembly
Figure 10 Shear stress from height versus yeast cell count.
Figure 11 Shear stress from flow rate versus yeast cell count.
Figure 12 Microneedle process.
Figure 13 Filtering step of the NANOLYSER device.
Figure 14 Mixing stage of the NANOLYSER device.
Figure 15 First heating stage of PCR.
Figure 16 Cooling stage of PCR.
Figure 17 Second heating stage of PCR.
Figure 18 Spectrophotometry stage of the NANOLYSER device.
Figure 19 Complete NANOLYSER device.
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Figure 20 Yeast cell adhesion in a microfluidics channel.
List of Tables
Table 1 Mesh details for the flow simulation
Table 2 Simulation conditions for a pressure difference of 49,000 dyne/cm2
Table 3 Simulation conditions for a pressure difference of 39,200 dyne/cm2
Table 4 Simulation conditions for a pressure difference of 29,400 dyne/cm2
Table 5 Experimental and theoretical values
Table 6 Relationship between Shear Stresses and Number of Yeast Cells
Table 7 Microfluidics statistical results
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1 Introduction
1.1 Problem Statement
Developed in the early 1980’s, microfluidics, the study of small volume flow through small
volume channels, has become an integral tool in the integration of engineering principles with
nanotechnology, which is an emerging science. Nanotechnology explores the development and
implementation of nano-sized devices to solve real world issues, and microfluidics techniques
and devices have many applications in this field.
Team X6 of the Fundamentals of Engineering Honors (FEH) Program at The Ohio State
University in the Nanotechnology Laboratory was tasked with conducting research to investigate
the factors affecting cell adhesion in a microfluidics channel, while simultaneously developing a
theoretical lab-on-a-chip NANOLYSER (Nano-functionalized Assay Nested in an Onboard
Laboratory Yielding Specific Expeditious Results) device utilizing different aspects of
nanotechnology. The first half of this report discusses Team X6’s observation of the effect of
surface topography on yeast cell adhesion in a microfluidics channel, while the second half
covers the creation of a custom NANOLYSER device capable of diagnosing cystic fibrosis with
a single drop of blood.
For the microfluidics study, Team X6 performed the necessary experiments and obtained
adequate data in order to discuss how cell adhesion on a surface can be affected by a change in
surface topography; specifically, how the presence of hexagonal microwells affects yeast cell
adhesion in a microchannel. The NANOLYSER design, on the other hand, is conceptual and
based off of information found from various pieces of literature.
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1.2 Team Introduction
This experiment was created and compiled by Turner Adornetto, Usoshi Chatterjee, Luke
Forshey, and Ryan Schoell (the team). All four students are participants in the FEH program.
The team was identified as “X6” for labeling purposes within the laboratory. The team met three
times a week during class to work on research related to both projects. Additionally, a minimum
of one to two meetings per week were scheduled outside of class. The team worked to reach
conclusive data on both of these topics through a process of research and experimentation.
1.3 Report Organization
The report is organized in the following sections indicated below:
1. Section 1 - Introduction: This section introduces the topic of research and the team working
on the research.
2. Section 2 – Microfluidics Background and Preliminary Concepts – This section presents a
brief overview of preliminary concepts of microfluidics. This section also discusses the
requirements and constraints of the project.
3. Section 3 - SolidWorks Simulation and Fluid Flow Program to Develop Microfluidics Design
Strategy – This section presents the SolidWorks simulation and the fluid flow program
developed using MatLab. Using these, the dependence of flow through the channel on
applied pressure was demonstrated.
4. Section 4 - Microfluidics Experimental Design Brainstorming and Preliminary Testing – This
section presents the development of the ideas for the chip assembly design and microfluidics
experimental design.
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5. Section 5 - Microfluidics Results and Analysis – This section presents the results obtained
from the microfluidics experiments and their analyses. This section also discusses the sources
of error.
6. Section 6 Microfluidics Summary and Conclusions – This section summarizes the work
done related to microfluidics and discusses future research.
7. Section 7 - NANOLYSER Design Background and Medical Application – This section
presents the medical application of the selected nanotechnology topic, and presents previous
work completed in the area. This also discusses the device structure, and the NANOLYSER
program.
8. Section 8 - NANOLYSER Strategy Development, Brainstorming, and Preliminary Design –
This section presents the preliminary design of the NANOLYSER and discusses the
development of the design.
9. Section 9 - NANOLYSER Final Design – This section presents the final design of the
NANOLYSER.
10. Section 10 - NANOLYSER Summary and Conclusions – This section summarizes the
critical components of the NANOLYSER, and discusses future research.
11. Section 11 – Bibliography
2 Microfluidics Background and Preliminary Concepts
2.1 Background
Microchannels can be defined as channels whose dimensions are less than 1 millimeter (mm),
and have high surface-to-volume ratio due to their small volume. Flow in microscale devices
differ from their macroscopic counterparts as the small scale makes molecular effects such as
wall slip more important, and it amplifies the magnitudes of certain ordinary continuum effects
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to extreme levels. As an example, strain rate and shear rate are proportional to the velocity and
inversely proportional to the length. So the shear rate experienced in a 10 micrometer (μm)
channel when the flow is 100 mm/sec is of the order of 104 sec-1. Fluids that are Newtonian at
ordinary rates of shear can become non-Newtonian at very high rates. The pressure drop in
microchannel flow can be very large, and since all of the work of the pressure difference against
the mean flow ultimately goes into viscous dissipation, effects due to internal heating by viscous
dissipation may be significant.
The flow of liquids through microfluidics channels usually occurs at low Reynolds numbers
(Re), which is the ratio of inertial to viscous forces. Re = ρVL/µ, where ρ is the density of the
liquid, L is a characteristic length of the system, in this case the height of the channels, µ is the
viscosity of the liquid, and V is the linear velocity of liquid through the channels. For systems at
low Re, Q, the volumetric rate of flow of liquid between two points in a channel, is proportional
to ΔP, the difference in pressure between two points, and can be written as ΔP ∞ Q. Using R as
the constant of proportionality, also known as the fluidic resistance, ΔP = RQ.
2.2 Preliminary Concepts
The microfluidics experiments focused on investigating the effect of shear stress on yeast cells
that were allowed to adhere to the inside wall of the microchannel. The chip was custom-made
using a polydimethylsiloxane (PDMS) solution of base and curing agent.
The cells of focus in this study were type 2 yeast cells provided by the FEH Nanotechnology
laboratory. Team X6 decided to use a seeding time of 20 minutes for each trial to allow
consistent and complete cell adherence to the channel walls. Once the cells have been allowed to
attach to the channel walls, water was passed through the chip by connecting the inlet tube to a
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syringe, which was placed at a height of 20 centimeters (cm) from the outlet tube. The
microchannel was observed under a microscope to determine the number of cells attached to the
channel wall. Changing this height of the syringe generated different shear stresses on the yeast
cells as the water flowed over them. The time allotted for the flush was maintained at exactly 5
minutes so that the shearing effect on the adhered cells was uniform. After each flush, the
microchannel was again observed under a microscope and the number of cells remaining
attached were compared with the number of cells originally present to determine the effect of
increasing shear stress on the adhered cells.
Experiments were based on the study conducted by Mercier-Bonin et al. (2004). Their study
investigated the attachment of microorganisms to a surface. Experiments were conducted with
yeast cells where shear-induced detachment of the yeast cells from a plane glass surface using a
specially designed shear stress flow chamber was studied. Experiments were conducted not only
with different yeast cell types, but also under different environmental conditions (ionic strength,
contact time). Surface physicochemical properties of the cells (surface charge, hydrophobicity,
and electron donor and electron acceptor components) were also determined. The results
obtained showed that the adhesion of hydrophilic cells were weak on glass plates, attributing this
to both electrostatic effects and hydrophilic repulsion. They also found that cells with higher
hydrophobicity adhered more strongly to glass. Results of this research could be used to predict
particles or biological materials that are able to attach themselves to various parts of the body in
various medical situations (hip replacement, prosthetics, etc.).
Further relevant research was conducted by Jiang (2011). In this instance, researchers
investigated the abilities of microfluidic whole-blood immunoassays to help with diagnostics
using a lab-on-a-chip device.
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2.3 Requirements and Constraints of Project
As stated previously, the microfluidics design project was focused on analyzing how the
presence of hexagonal microwells in a microfluidics channel affected cell adhesion on the walls
of the microchannel under varying shear stresses. In order to consistently measure this, it was
pertinent to adequately understand the components of flow through a microfluidics channel.
Water was chosen to be the fluid of focus, based on cost, supply, and predictability, while the
mode of fluid delivery was assumed to be continuous-flow.
Continuous flow was implemented with an elevated water source; thus, creating a high pressure
inlet. Additionally, in order to create consistency amongst trials, it was also necessary to induce
laminar flow through the channels. Laminar flow occurs when a fluid flows in layers, with the
fastest velocity occurring at the center of the channel and the slowest velocities at the outer edge.
The laminar flow was tested and quantified through simulation and preliminary testing with
SolidWorks Flow Simulation, a Microfluidics Program, and a testing chip, which was provided,
in order to justify its existence in the flow through the microfluidics channel.
3 SolidWorks Simulation and Fluid Flow Program to Develop Microfluidics Design Strategy
3.1 SolidWorks Simulation
The first step in effectively simulating fluid flow through a custom-made microfluidics channel
was to construct the channel in SolidWorks. To do this, simulations were conducted with a
channel that matched the dimensions of the microfluidics channel of focus. The mesh itself is
shown in Figure 1 (Figures Appendix) while the mesh details specified for this simulation can be
found in Table 1 (Tables Appendix). Global goals of average and maximum velocities were
chosen in order to observe convergence of the simulation, while assumptions disregarding the
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inclusion of temperature, mean radiant temperature, operative temperature, heat flux, and heat
transfer rate of the fluid were made for ease of calculation. Evidence of convergence is
exemplified in Figure 2, as the lines representing maximum and average velocity of fluid through
the channels approaches a specific value for a pressure head of 49,000 dynes per square
centimeter (dyne/cm2).
The SolidWorks solver was then implemented to perform calculations on the channel and mesh
for three separate pressure differences: 49,000 dyne/cm2, 39,200 dyne/cm2, and 29,400 dyne/cm2.
At each of these pressure differences, a goals plot of maximum and average fluid velocity
through the channel was obtained. Similarly, multiple plots were constructed for the channel in
order to gain further information pertaining to the flow. Cross-sectional velocity contour cut
plots at the testing region (the area in which cell adhesion was analyzed) were made.
Furthermore, a shear stress surface plot was created. Each of these on-channel plots (goals plot,
velocity contour, and shear stress) were made for each pressure difference, however, only the
goals plot, velocity contour, and shear stress plot for a pressure difference of 49,000 dyne/cm2
(Figure 2 through Figure 4) are shown. A table of values for each pressure difference was made
(Tables 2, 3, and 4) and include the following values pertaining to the conditions of each run:
inlet pressure, outlet pressure, density, viscosity, number of iterations, number of iterations per
travel, delta value, maximum velocity in the testing region, and wall shear stress.
In the experimental testing of the channel, flow was assumed to be laminar. This assumption was
supported by the SolidWorks simulation, specifically, by the velocity cut plots in the testing
region of the channel. For example, the cut plot in Figure 3 can be drawn upon to analyze this
claim. For this trial, and all of the other trials as well, the plot shows that the largest fluid
velocities through the channel occur at the center of the channel’s cross section. Similarly, the
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smallest velocities are shown to occur near the edges of the channel. These observations are
concurrent with observations drawn from un-assumed laminar flow, and thus, the simulated flow
assumption of laminar flow was supported. Later, the importance of laminar flow will be
discussed.
3.2 Fluid Flow Program
The microfluidic program created on MatLab was used to find the shear stress in the channels
based on channel dimensions like height, width, and length of the channel as well as changes in
pressure, flow rate, and viscosity. The height of the channel was 0.2 cm, the width of the channel
was 3 cm, the length of the channel was 25 cm, the change in pressure from inlet to outlet was
1000 dyne/cm2, and the viscosity of the water was 0.01 g/cm-s. Flow rate was determined
through experimentation.
The results obtained in this program coincide with the material found in literature. However, a
way to physically prove these simulation results would be to gather a pressure-fluid velocity
relationship through actual testing with the custom made microfluidics channel. With this data,
many other values pertaining to the experiment, such as Reynolds number, shear stress, and
others, could be compared with those gathered with the simulation. Fortunately enough,
experimental data does, in fact, exist relating to this computer program simulation. The trends
observed experimentally match the trends observed in this simulation, specifically, as pressure
increases, so does the flow rate, or velocity through the channel, and the shear stress experienced
by the channel. The fluid mechanics program itself is pictured in Figure 5.
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3.3 Flow Dependence on Applied Pressure
Experiments were conducted to observe the dependence of flow in a microfluidic channel on
applied pressure. Experimental data collected was compared against theoretical data obtained
using SolidWorks Flow Simulation and Fluid Flow Program. The data obtained were used to
create a calibration curve of flow rate versus pressure for one of the channels on a standard-
design chip.
3.3.1 Results Obtained
Due to complications resulting in the construction of the microfluidics channel tested in the
experiment, sample flow rate measurement data for three trials, along with the channel and fluid
characteristics, was provided so that proper analysis could be conducted. With this data, hand
calculations were performed in order to determine the theoretical flow rate, theoretical pressure,
and mean theoretical flow rate, and Table 5 presents the experimental and theoretical values.
Furthermore, a relationship between theoretical flow rate and water column height was created
and is shown in Figure 6.
Using the fluid mechanics program developed, the theoretical flow rate along with the channel
dimensions and fluid characteristics were implemented in order to determine the change in
pressure, average velocity, Reynolds number, and shear stress at wall for each trial. In addition to
the values mentioned above, the theoretical flow rate was then calculated using the theoretical
pressure. With values for theoretical pressure and theoretical flow rate, two additional plots were
constructed: Figure 7 contains the plot of theoretical pressure versus water column height and
Figure 8 contains a plot of theoretical flow rate versus theoretical pressure.
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3.3.2 Discussion
In order to gain further information pertaining to the flow through microfluidics channels,
SolidWorks was used to perform flow simulation sensitivity analysis on channels of varying
cross sectional dimensions. With the rectangular channel used in Lab 1, two additional channels
were constructed by varying the height by 5 percent. Using an experimental pressure of 9949
dyne/cm2 as the inlet condition, surface plots and surface parameters showing the contours of
wall shear stress on the bottom of the channels were created.
The relationship between flow rate and height (Figure 6) can best be described as a positive
linear relationship. The flow rate increased as the height of the water column increased. Figure 7
shows a relationship between pressure and height of the water column which can also be
described as a linear positive relationship. Figure 8 depicts a relationship between flow rate and
pressure which is a positive linear relationship for both experimental flow rate vs. experimental
pressure and theoretical flow rate vs. experimental pressure. As pressure increased both
theoretical and experimental flow rate increased.
Some reasons why experimental flow rate might be different from theoretical flow rate are
because theoretical flow rate assumes constant variables when in reality not every variable can
be held constant. Also the theoretical model assumes perfect geometry of the channels when in
reality the channels are not perfect and can lead to changes in flow rate. Another reason why
theoretical flow rate might differ from experimental is because the theoretical model assumes no
interaction between the water and the walls of the channels besides shear stress when in reality
there are other forces such as adhesion that can affect the flow rate. Also air bubbles that might
have gone through the channels could affect the flow rate.
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Some assumptions that were necessary to calculate the flow rate using the fluid mechanics
program were constant flow rate, constant change in pressure, and constant viscosity. The
assumptions made in order to use the program were generally good because these variables
should not have changed much so the program should produce data that is similar to the results.
If a nanodevice were being used to separate out ionic species, the flow profile should be constant
as in EOF with a thin double layer because a good way to separate ions is using and electric field
which causes a constant flow profile. The positive cations will flow towards the cathode while
the anions will flow towards the anode.
A positive finite velocity at the wall should lead to a greater flow rate than if the velocity at the
walls was zero because the water at the wall with zero velocity is slowing down the particles
near it which slows down the particles in the center. The velocity at the walls that is positive has
less effect on the particles near it which in turn has less effect on the particles in the center.
3.3.3 Summary and Conclusions
Based on the experimental data, the flow rate was found to directly proportional to the height of
the water column, and since the height of the water column is also directly proportional to the
applied pressure, the flow rate is directly proportional to the applied pressure. Any difference
between the theoretical flow rate and the experimental data can be attributed to the fact that all
variables affecting the flow rate other than the applied pressure could not be kept constant during
experimentation. Also the theoretical model assumes perfect geometry of the channels when in
reality the channels are not perfect and can lead to changes in flow rate. Deviations from the
theoretical value can also be due to presence of air bubbles in the flow channel, though effort
was made to avoid air bubbles.
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A positive finite velocity at the wall should lead to a greater flow rate than if the velocity at the
walls was zero because the water at the wall with zero velocity is slowing down the particles
near it which slows down the particles in the center. The velocity at the walls that is positive has
less effect on the particles near it which in turn has less effect on the particles in the center.
3.4 Decision Making Process
Based on the SolidWorks flow simulation, the design for the chip was a straight channel with a
depth of 130 µm. The flow simulation showed the shear stress along the walls of the channel
with this design, and also showed that the velocity profile is parabolic shaped. The standard chip
calibration also showed that a depth around 130 µm would produce a good flow and showed a
reasonable increase in flow rate as height was increased. The chip was also designed to have four
equal channels so that multiple trials can be done more efficiently. Also, having four parallel
channels made it easier to observe the channels under the microscope and decipher which
channel is the channel with yeast cells in them.
4 Microfluidics Experimental Design Brainstorming and Preliminary Testing
4.1 Brainstorming
The microfluidics experiments involved taking a chip with channels that are micrometers deep
and experimenting with cell adhesion of yeast cells. Initially a yeast-water suspension was
allowed to pass through the channels and the yeast cells attached to the walls of the
microchannel. Water was then allowed to flow through the chip under varying shear stress
conditions. Shear stress was changed through changing the height of the water column.
Application of the shear stress on the attached yeast cells detached them from the walls, and the
number of yeast cells remaining were observed under a microscope. Difference in the cell
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number before and after water was allowed to flow provided the number of yeast cells that were
sheared off under a particular shear stress.
4.1.1 Chip Assembly
Prior to conducting the experiment, the microfluidic chip was assembled. There are many
components that make up the microfluidic chip. This includes a chip top which was made out of
PDMS and had the micrometer channels engraved in it. Also the chip top had inlet and outlet
ports with holes through them to allow water in the channel. The chip bottom was also made out
of PDMS and was put on the channel side of the chip top to create a wall that kept in the water
and the yeast cells. The top part of the chip holder, which was acrylic, had holes to allow for
plastic adaptors attached to tygon tubes to be inserted into the chip. The top part of the chip
holder had other holes that lined up with holes of the bottom part of the chip holder that allowed
screws to be put through them to squeeze the chip top and bottom together and to hold them
together. Tygon tubes are used to transport water into and out of the chip.
Understanding the constraints of the geometry of the chip and chip holders were necessary so
that proper testing and fabrication could be conducted within the FEH Nanotechnology
Laboratory. When creating the components of the chip, chip holder, and wafer discussed
previously, size benchmarks had to be met. The wafer, chip top, and chip bottom had to be
cylinders with diameters of 2 inches and depths of 0.125 inches. Alternatively, the chip holders
were to have a depth of 0.25 inches with the cross sectional shape unique to Team X6’s design.
For the wafer and chip top, accurate channel geometries had to be reflected in the designs. The
depth of the channels had to be between 120 and 150 micrometers, while the width of the
channels had to be between 300 and 400 micrometers. By the nature of the fabrication equipment
used in the chip holder, specifically, a laser cutter, a kerf of 0.004 inches had to be accounted for.
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The fabrication of the wafer had a similar stipulation. The wafers were created by milling the
material away, thus, the interior curves of the design could not be less than the size of the bit
(1/64 inch) and the separation of parallel interior edges could not be less than the diameter of the
bit (1/32 inch).
Furthermore, minor additions and adjustments were made to uphold the functionality of the chip.
For example, a testing region were marked on the chip top, the channels could not be too close to
the edge of the chip, only one depth could be milled, so a chip with varying depths is not going
to work. Finally, team identification was to be included on the chip top.
As part of the initial brainstorming, the chip was designed in such a way so that the lengths of
each channel would be the same and would be parallel to each other. This was done in order to
be able to test cell adhesion quickly using multiple channels. Some of the initial brainstorming
that led to design of the chip holder included having a triangle shaped holder with holes along
the base and holes that were parallel to them that were about the length of the channels on the
chip apart to allow for easier chip assembly.
4.1.2 Chip Design Changes
Furthermore, minor additions and adjustments were made to uphold the functionality of the chip.
For example, a testing region must be marked on the chip top, the channels cannot be too close to
the edge of the chip, and only one depth can be milled, so a chip with varying depths is not going
to work. Finally, team identification was to be included on the chip top.
4.1.3 Microfluidics Final Design
In the final design, the diameter of the chip top remained at 5.08 cm, with four equal and parallel
channels that are 130 µm deep. The chip bottoms were varied between one with no patterns and
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another with hexagonal wells. The chip holder is an equilateral triangle design that has holes for
the ports along the base and more holes for ports that are parallel to the holes along the base and
these are set apart about the distance of the channels on the chip. Three holes are located at the
corners of the triangle of the both the top and bottom part of the chip holder to allow screws to be
put through them.
There are two parts to a chip, the positive and the blank base. Since the variable that we are
trying to test is the effect of the texture of the bottom part of the chip on yeast cell adherence, a
chip base with hexagonal wells was used. Figure 9 shows an exploded view of the chip, chip
holder assembly.
4.2 Microfluidics Experimental Design
4.2.1 Hypothesis
For the present research in microfluidics, the null hypothesis was that hexagonal microwells will
not affect cell adhesion in a microfluidics channel. In order to adequately reject or not reject the
above null hypothesis, an alpha-level of 0.05 was chosen, or the confidence level was set to 95
percent. The procedure followed in order to gather sufficient data to support or reject the null
hypotheses will now be described. The alternate hypothesis was that channel geometry will have
an effect on cell adhesion.
4.2.2 Experimental Procedure
The purpose of the experiment was to test how shear stress affects yeast cell adhesion to a
channel surface. Appendix A presents the experimental procedure. At the start of the experiment,
a mold using PDMS was prepared from a designed chip mold negative. The PDMS was first
made from 1.47 milliliters (mL) of curing agent and 13.97 mL of base to make 15.44 mL of
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PDMS according to the ratio of curing agent to base. This mixture was then poured into a petri
dish, with a diameter of 2 inches. This petri dish became the standard chip base. Additional
amount of the PDMS mixture which was about 7.72 mL was poured into another 2 inch diameter
petri dish with the designed chip mold negative. Both petri dishes were then put into a desiccator,
which was connected to a vacuum pump. The air bubbles in the PDMS were pulled to the surface
by pressure and were removed by increasing pressure to break the bubbles. After the bubbles
were removed, the petri dishes are put into an oven at 95 ºF for 20 minutes to cure. Once the
PDMS mold cured, the base chip was demolded from the petri dish, and the top chip was
demolded from the chip design mold negative and the petri dish.
A water yeast mixture was prepared for the experiment. Initially, using a digital scale, the mass
of an empty beaker was recorded. The scale was then set to zero, and approximately 2 grams of
yeast was added to the beaker, followed by the addition of 10 mL of warm 150 mM saline
solution using a 3 mL syringe. The contents in the beaker were thoroughly mixed and allowed to
incubate in the beaker for 20 minutes.
As the yeast was incubating in the beaker, the microfluidic testing was set up. After procuring 10
inch and 6 inch Tygon tubes, one end of each tube was attached to a clear adapter and a green
tip. The other end of the 10-inch Tygon tube was attached to a 20 mL syringe filled with water,
and was taped to a wooden tower situated 35 cm from the ground. The other end of the 6-inch
Tygon tube was attached to the wooden tower at a height that allowed the end of the Tygon tube
with the clear adapter and green tip to reach the area where the PDMS chip would be located.
This end of the 6 inch Tygon tube was placed above a plastic cup to collect water during the
microfluidics experiment.
16
At the beginning of the experiment, mass of the empty plastic cup was recorded on an electronic
scale. After the yeast water mixture in the beaker has incubated for 20 minutes, a 1 mL syringe
was used to take a sample of the yeast water mixture and inject it into the channels of the chip.
This was done by inserting the syringe into the entrance port of the chip and injecting the water
yeast mixture until the mixture starts to come out of the exit port of the chip. Flow was not
induced for 20 minutes to allow the yeast to adhere to the walls of the channel. After 20 minutes,
the green tip attached to the 10-inch Tygon tube was inserted into the entrance port of the chip
and the green tip attached to the 6-inch Tygon tube was inserted into the exit port of the chip.
The clamps on both Tygon tubes were then removed and the yeast cell were flushed for
approximately 5 minutes. After 5 minutes, the mass of the cup with water at the end of the 6-inch
Tygon tube was measured, and the mass of the water collected was obtained upon subtracting the
mass of the empty cup. The plastic cup was then emptied, dried, and placed back under the open
end of the 6-inch Tygon tube.
The channel with the yeast cells was then observed at a specific part of the channel, marked
using a Sharpie, under a microscope using a magnification of 100X. A picture of the channel
under the microscope was taken using a camera and the number of yeast cells was counted.
For this experiment, two different chip bases were used. One was patterned with hex wells and
the other was unpatterned. The hex wells were 5 µm in depth, 10 µm in diameter, and 34 µm
apart from each other. The channel in which each experiment was performed in was recorded to
accurately judge the effects of certain channel geometries.
The process of subjecting the chip to flow followed by observing the channel under the
microscope was repeated at heights of 45 cm, 55 cm, and 65 cm. This was counted as one data
17
point and four more were obtained using the standard base chip with no pattern, and then five
data points were obtained using the patterned chip base with hex wells. Hex wells are hexagonal
cut-outs in the bottom of the channel. They are 5 µm deep, 10 µm in diameter, and spaced 10 µm
apart in a square pattern. If at any time the channel showed clumps of yeast cells or yeast cells
still flowing in the chip under the microscope, an additional 2 minutes of flow was induced.
5 Microfluidics Results and Analysis
5.1 Results Obtained
Table 6 presents the values of microchannel dimensions, number of yeast cells after flow, flow
rates, pressures from flow rate and liquid height, and the corresponding shear stresses. Figures 10
and 11 demonstrate the relationships between shear stress from height with cell count and
between shear stress from flow rate with height and cell count. Figure 20 shows a picture of
yeast cells adhered to the channel wall.
5.2 Discussion
Liquid height was varied to change the applied pressure and the shear stress on the attached yeast
cells. As shown in Figure 10, number of yeast cells attached to the walls of the microchannel
decreased with increase in shear stress due to variation in liquid height (τH). As discussed in the
previous section, three data points were collected, and for each data point four sets of readings
for applied shear stress and corresponding yeast cells attached to the surface were collected. A
best-fit line was constructed for the first data point using Microsoft Excel, and it was found that
the power function generated a line that best described the trend. The equation of the best-fit line
was found to be
y=2×106 x1.804
18
with y as the number of cells attached and x is the applied shear stress due to liquid height. The
R-squared value was found to be 0.7795, and demonstrates how close the data fitted the
regression line. It is also known as the coefficient of determination, or the coefficient of multiple
determination for multiple regression.
Based on experimental observations and class discussion, a parameter called “threshold yeast
shearing” was defined as the condition when approximately 70 percent of the attached yeast cells
were dislodged due to the applied shear stress. For the first data point, if 70 percent of original
yeast cells were sheared off, then the number of yeast cell that remained attached would be
approximately 202, and using the above regression equation, the shear stress value would be
163.8 dynes/cm2, and this would be value for threshold shearing. This method for finding
threshold shear stress is not entirely dependable since the R squared constant is only 0.7795. In
future experiments, if threshold shearing is defined before any experiments, and then try to shear
just that percent of cells away, then more accurate results should be collected.
Shear stress is also dependent on flow rate (τQ) and number of yeast cells remaining attached to
the walls of the microchannel as a function of shear stress from flow rate were plotted in Figure
11. Three data points were plotted as indicated by the three sets of data, though the best-fit line
was constructed for the first data point only. A best-fit line was constructed for the first data
point using Microsoft Excel, and it was found that the second-degree polynomial function
generated a line that best described the trend. The equation for the best-fit line was found to be
y=66985 x2−14213 x+900.07
with y as the number of cells attached and x is the applied shear stress due to flow rate. The R-
squared value was found to be 0.9894. With this equation, the interpolated value of threshold
19
shearing was found to be 0.07708 dyne/cm2. This method is a good approximation for the actual
value since the R-squared value is 0.9894.
When interpolating data, it is better to compare the cell count to the shear stress calculated from
the flow rate. Since human error is always present, the height and flow rate values already have
some error in them. When the shear stress is calculated from the liquid height, error is higher as
the shear stress is dependent on height raised to the power of 3. Taking the cube of a variable
with error in it magnifies the errors in the result. However, shear stress is dependent on the flow
rate value only, and the shear stress calculated from flow rate is more reliable. This is evident by
the difference in R squared values of the best-fit equations.
A reverse flush may be necessary if the entrance port is clogged with yeast cells. The reverse
flow would force the cells out of the way. Another reason a reverse flush may be necessary
would be to get rid of yeast cells that are stacked on top of each other inside the channel. If there
were yeast cells resting on top of others, then they would not adhere to the surface and would
give erroneous data.
The advantage of allowing the yeast to incubate for longer periods of time include the ability to
observe how quickly yeast adheres to the surface and also the possibility of getting more cells
within the sample range. Some of the disadvantages of longer incubation period are running a
greater risk of clogging the channel and risking cells adhering to the top and sides of the channel.
In addition, if the yeast cells are allowed to incubate for a longer duration, it may be difficult to
flush them through the channel.
Some design features that can mitigate issues with the yeast cells are a wide inlet and a smooth
channel surface. If the inlet is wider, it will be easier to drop the yeast cells right on the entrance
20
without clogging up the whole opening. Additionally, if the channel surfaces are smooth, there
are less bumps and dents for the yeast cells to latch on to.
It is unlikely that surface irregularities on the inside of the channel would affect the flow. Any
error caused by surface irregularities would likely be random rather than systematic, since bumps
or dents would induce turbulent flow. It is anticipated that the flow rates in the channel with
yeast cells in it will be slower than the flow rate in an empty channel. This is because some of
the momentum in the water will have to be used to overcome the inertia of the cells to shear
some of them away.
It would be possible to run such an experiment if multiple channels are present. The
experimental procedure would have to be modified to have each inlet and outlet fastened in an
organized way to allow liquid to travel in each of them simultaneously, as well as having a way
to clamp and unclamp each tube at the same time.
It has been assumed that the conditions for all of the channels are the same because they were all
designed and made identically. Although, if there were a defect in one of the channels, it would
clearly have an impact on the results gathered from that channel. It could be verified that they
are the same by having water flow through each of them and comparing the flow rates of each
channel.
In the beginning, it was difficult to get water to flow through the chip. However, a few of our
members would go to open labs and collect data. In the end, enough good data was collected to
call the experiment a success.
21
It is recommended fastening the inlet and outlet tubes to stationary objects instead of holding
them by hand. This would eliminate some human error and make the process easier for the
experimenters.
The threshold shear heights seem to be fairly repeatable. Every data point had the threshold
shearing occur at the third height that was tested. However, the drop below the threshold yeast
shearing was much more dramatic for the second data point. This may be due to some yeast
clumping up in the channel.
The percent differences from the average in the calculated shear stress from flow rate varied
between -10% and 13%. This large range is likely because the flow rates differed greatly
between data points. All of the percent differences from the average in the calculated shear stress
from height are 0%. This is simply because the same heights were chosen for each data point.
Compared to the graphs reported in the Mercier-Bonin paper, our graphs have the same general
shape. On both graphs, the two variables are clearly inversely related. The biggest difference is
that this research project reported the actual number of cells after each shearing, whereas the
Mercier-Bonin paper reported the percent of cells sheared away.
5.3 Summary and Conclusions
At the end of the experiment, relationships between the cell count and shear forces for both
height and flow rate were derived, allowing for the determination of the threshold yeast shearing
value. Though the same data set was used to calculate the shear stresses from height and from
flow rate, there were no deviation (0%) in the case of shear stress from height and the deviation
ranged from -10% to 13% in the case of shear stress from flow rate. Overall, the data reported
22
shows a fairly high degree of central tendency, along with mirroring the graphs reported in the
Mercier-Bonin paper, in turn, validating the results concluded upon.
While the information found within the experiment was vital, the knowledge gained in
conducting the yeast culturing and shearing experiment was most valuable. Information
pertaining to the set-up, procedure, and analysis is essential to properly executing future flow
experiments.
5.4 Calculations and Mathematical Analysis
Sample CalculationsQ=V/t
Q=(2.20 mL)/(300 seconds)Q=0.0073 mL/s
Q-Flow Rate (mL/s)V-Volume of water (mL)
t-Time (seconds)
ΔP=((12)(Q)(U)(L))/((H3)(W))ΔP=((12)(0.0073 mL/s)(0.01 g/cm-s)(25 cm))/((0.23 cm)(3 cm))
ΔP=0.91667 dynes/cm2
ΔP-Change in Pressure (dynes/cm2)Q-Flow Rate (mL/s)U-Viscosity (g/cm-s)
L-Length of Channel (cm)H-Height of Channel (cm)W-Width of Channel (cm)
T=(ΔP)(H)/((2)(L));
T=(0.91667 dyne/cm2)(0.2 cm)/((2)(25 cm))T=0.0018333 dyne/cm2
T-Shear Stress (dynes/cm2)ΔP-Change in Pressure (dynes/cm2)
H-Height of Channel (cm)L-Length of Channel (cm)
AV=(DP)(H2)/((12)(U)(L))
23
AV=((0.91667 dynes/cm2)(0.2 cm)2)/((12)(0.01 g/cm-s)(25 cm))AV=0.012222 cm/s
AV-Average Velocity (cm/s)ΔP-Change in Pressure (dynes/cm2)
H-Height of Channel (cm)U-Viscosity (g/cm-s)
L-Length (cm)
Re=(AV)((4)(W)(H)/((2)(W)+(2)(H))/U)Re=(0.012222 cm/s)((4)(3 cm)(0.2 cm)/((2)(3 cm)+(2)(0.2 cm)/0.01 g/cm-s)
Re=0.458333Re-Reynold Number
AV-Average Velocity (cm/s)W-Width of Channel (cm)
H-Height (cm)U-Viscosity (g/cm-s)
Le=(0.06)(Re)(4)(W)(H)/((2)(H)+(2)(W))Le=(0.06)(0.458333)(4)(3 cm)(0.2 cm)/((2)(0.2 cm)+(2)(3 cm))
Le=0.0103125 cmLe-Entrance Length (cm)
Re- Reynold NumberW-Width of Channel (cm)H-Height of Channel (cm)
5.4.1 Results for Flow Rate and Shear Stress for Each Base Type
From the procedure detailed above, data relating to flow rate and shear stress was gathered.
These results show the effect of channel geometry, as detailed below.
As the height of the water column was increased, the channel experienced a greater shear stress.
The average shear stress of the unpatterned surface was 2.6*10^2 dynes/cm^2, whereas the
average shear stress of the patterned surface was 2.45*10^2 dynes/cm^2. This shows that the
patterned surface took a slightly higher shear stress to shear the cells away.
24
5.4.2 Results in Terms of Hypothesis
The surface of the microchip played a huge role in the simplicity and effectiveness of detecting
the percentage of yeast cells left in the flow channel. As mentioned previously, the patterned
surface was much more effective in keeping the yeast cells inside of the channel. This is due to
the design of the patterned surface: the wavy pattern did not allow as much laminar flow as the
smooth surface, and the uneven surface kept more yeast in the channel than the flow channel.
The downside to using this design is that the height of the water column must be much greater
than for the smooth surface.
The null hypothesis the team came up with was that surface topography will have no effect on
yeast cell adhesion, and the alternative hypothesis was that surface topography will have a
statistically significant effect on yeast cell adhesion. An alpha level of 0.05 was picked, mainly
for the reason that it was a very standard value chosen for type 2, 2-tailed t-tests. A statistical
analysis was performed to decide whether the results were statistically significant. With α value
of 0.05, a type 2, 2-tailed t-test was performed on the data, and the p-value from the t-test was
0.977 (Table 7).
Based on shear stress calculations and using a 2 tailed t-test, no statistically significant difference
was observed between the shear stress needed to dislodge cells from the patterned hexagonal
welled surface and the blank chip, as the p-value (0.977) was greater than α/2 (0.025). Null
hypothesis was not rejected, and surface topography does not significantly affect cell adhesion.
Since the average diameter of the yeast cells (Saccharomyces cerevisiae) used in the experiment
was 5 to 10 µm, it is possible that the 8 µm wells on the patterned surface could not
accommodate the larger yeast cells; thus, reducing the ability of the cells to adhere. Conclusions
are applicable to the particular species of yeast cells tested and patterned surface used for the
25
experiment. For a more generalized conclusion, the study needs to be expanded to include a
larger, more diverse range of patterned surfaces, variety of yeast cells, and significantly more
data points.
5.5 Sources of Error
5.5.1 Error within the Experiment
There were a few sources of error present in both parts of the testing. The prime source of error
was leakage in either the entrance ports or exit ports of the channel. This was due to green Tygon
tips that were either placed too far in the port or too loosely in the port. Leakage caused an
incorrect volume of water to be recorded due to the water that was not accounted for during the
weighing of the plastic cup. This caused other discrepancies in the calculated values as each
calculation required the volume of water as one of its variables. Another source of error in the
testing was yeast being clogged in the flow channel. Though the pressure of water in the channel
would eventually cease the blockage of yeast, this did not solve the lost amount of water that was
transferred into the plastic cup. Because the trials each had a set time, the amount of water in the
cup at the end of a trial where yeast blocked the flow of water was lower than normal. This
caused the exact same problem with calculations as the errors associated with leakage.
5.5.2 Experimental Error
There were some errors in the experiment. For some of the trials, there was a small bubble that
got into the channel. This caused an excessive amount of yeast to be sheared away on the first
trial and in turn yielded erroneous results.
Another error in the experiment was the time allowed for the yeast to adhere to the channel. If
the time allowed was too long, then the shearing would not be as effective, and if the time
26
allowed was too short then the shearing of the yeast would occur faster than it should have. The
yeast at one point was left in the channel for a shorter time than desired which created false data
since the shearing occurred at a more rapid pace than expected. Ideally, the experimental
pressure would match up with the calculated theoretical pressure using the experimental flow
rate and other known parameters. Similarly, the experimental flow rate would be the same as the
theoretical flow rate that was calculated using the experimental pressure. This was not the case
because the data that was experimentally determined for the flow rate and pressure were not
perfect for the parameters given and therefore did not exactly match up to their theoretical
values. This could be due to equipment error such as cracks in the chip, or due to human error
such as misreading the amount of water injected into the tube or not correctly recording the time.
The equipment error would create a determinate error that would affect the data uniformly, while
the human error would affect the data randomly because it would be an indeterminate error.
6 Microfluidics Summary and Conclusions
6.1 Summary
The experiments conducted regarding the microfluidics aspect of this research project analyzes
the effect of hexagonal microwells on cell adhesion within the channels themselves. To test this,
two control groups were established: first, a channel was calibrated by having no yeast cells and
measuring the undisturbed flow of water through the microchannel; and a second where cell
adhesion was measured in an unpatterned channel (no microwells). Then, a microchannel
containing hexagonal microwells was tested. Five data points containing three trials each were
gathered for the two control groups and one variable group. The heights measured stayed
constant throughout the trials along with the cell seeding time when yeast cells were used. Once
27
completed, the analyzed data was used to make determinations on the microwells’ effect on cell
adhesion.
6.2 Roadmap for Future Work
While the experiments performed to analyze the proposed hypotheses were fairly accurate, there
were still errors within the experimentation. The dimensions of these tips could be calculated to
fit into the entrance and exit ports more precisely and allow no space for any water to escape.
Also, a wider channel may prove to increase accuracy due to a lower chance of clogging. This is
debatable however, as yeast clogging happens all the time and is almost bound to happen once
every couple of trial runs.
Other ways to improve upon this experimentation would be to create a holder with a viewing
hole that can be used during patterned chip testing, and testing more patterned surfaces. The chip
holder with the viewing hole made the yeast cells far clearer in the pictures obtained through the
microscope. The pictures taken using the chip holder top without the hole were not clear
(blurred), and therefore, it was difficult to obtain an exact count of yeast cells in the channel. If
another type of hole was created to create enough pressure over the entire chip to keep the
patterned surfaces sealed off from leakage, clearer pictures could be taken and a more accurate
definition of significant shearing could be used in experimentation.
Along with these corrections to the experimentation, this testing could pave the way for future
experiments. For example, the same type of testing could be run using these designs, but with a
different fluid other than water. The purpose of this would be to see how a fluid with the same
density as say, a bodily fluid like blood would act in the same experiment. Liquids with different
28
viscosity should also be used for experiments, as viscosity would cause a change in flow rate and
also would most likely alter the shearing of the yeast cells.
Further research would also be beneficial for monitoring yeast adhesion by having a base that
was half patterned and half smooth. Running a parallel experiment would be beneficial because
the experiment could happen in less time. This would allow analysis to be performed at the same
time and would allow less experimental error since the shearing of the yeast could be monitored
at the same time under a microscope. Finally, increasing the number of tests could expand the
research for this experiment. The more tests performed would only make the data more valid.
7 NANOLYSER Design Background and Medical Application
Nanotechnology has become one of the most quickly developing fields of research in the
development of medical devices. The demand for more efficient and cheaper methods to analyze
blood samples, along with a high demand as a result of an aging population, had led to an
increased need to develop something that can better meet these needs. The field of diagnostic
nanotechnology is so new that there is much room for development and exploration in the
designs and use of these chips.
7.1 NANOLYSER Background and Chosen Medical Application
Our NANOLYSER device is a research based project. The goal is to theoretically design a lab
on a chip that will be able to detect a particular disease with one drop of blood from the patient.
The research focused on diagnosis of cystic fibrosis (CF). CF is a genetic disorder that affects
the lungs and digestive system of about 30,000 children and adults in the United States (70,000
worldwide). A defective gene and its protein product cause the body to produce unusually thick,
29
sticky mucus that clogs the lungs and leads to life-threatening lung infections; and obstructs the
pancreas and stops natural enzymes from helping the body break down and absorb food. This is a
genetic disorder that can be traced to the cystic fibrosis transmembrane conductance regulator
(CFTR) protein.
The chosen medical application for the NANOLYSER design was to be able to diagnose both a
carrier and a person with for cystic fibrosis. This disease has a specific mutation in its genome
and is a disease that calls for a quicker and less painful diagnosis. The objective of the carrier
testing by the NANOLYSER will be to conduct a laboratory test done on a sample of blood or
saliva to see if a couple is at risk for giving birth to a child with CF. Carrier testing is not
infallible. It cannot detect all of the CF gene mutations. In rare cases, a person can have a normal
test result and still be a CF carrier. The importance of carrier testing is that if CF is caught at an
earlier stage, a more regimented lifestyle can be followed from an earlier age, allowing for
greater coping normally. The NANOLYSER will test if a couple is at risk for giving birth to a
child with cystic fibrosis.
The design of the chip will be such that it will be able to break the cells and nuclear membranes
of the cells and detect whether the CFTR gene is present. Simultaneously, the chip should be
able to filter away any materials that could affect the results. Design of the NANOLYSER is
based on research made on cell separation (Helene Andersson 2003). Cell separation is important
in detecting some diseases because some diseases only need the white blood cells to determine if
a disease is present so separating the white blood cells from the red blood cells is important.
30
7.2 Brainstorming
In order to detect the CFTR gene, the NANOLYSER device had to contain a multitude of
preceding steps. Ideally, the blood sample would be extracted painlessly, possibly through the
implementation of a microneedle. Figure 12 below shows the microneedle process. Then, the
CTFR gene would need to be isolated and manipulated for easy and accurate cystic fibrosis
determination. To do this, the white blood cells within the sample would need to be lysed and
separated from the red blood cells in the sample. A series of heating and cooling steps would
follow, inducing polymerase chain reaction (PCR), allowing for a spectrometer to more easily
detect the CFTR gene.
7.3 Device Structure
7.3.1 Testing Designs
While the NANOLYSER device is theoretical by nature, real-world processes and technology
were implemented in order to develop a device that would be both accurate and reliable if ever
created.
Thus, to adequately prepare the sample for a determination of cystic fibrosis, processes including
extraction, filtering, mixing, PCR, and spectrophotometry was necessary. The theoretical design
of each step is described below.
A microneedle connected to the NANOLYSER channel pricks the finger of a patient and
funnels a small sample of blood into the device. From this point forward, electroosmotic force
controls the motion of the sample.
This mixture then passes through a filter made of microscale columns. Each column is 80
micrometers in diameter, spaced away from the nearest column 88 micrometers from center to
31
center. This leaves a space approximately 8 micrometers wide for the sample to pass through at
any given point. Since the average red blood cell is from 8 to 10 micrometers in diameter, the
filter will allow the plasma and white blood cell insides to pass through, keeping the red blood
cells behind. The filter is pictured in Figure 13.
The sample then flows through a mixing section of the channel. Small barriers etched in the
bottom of the channel were constructed to create turbulent flow through the channel. This step
allows an even distribution of components throughout the mixture, a vital step for latter
processes. The barriers are pictured in Figure 14.
From the mixing channel, the sample is passed into a heating chamber, the first chamber of the
PCR process. PCR allows rapid production of short pieces of DNA, even when nothing more
than the sequence of the two primers is known. During PCR, large amounts of pure DNA is
made available, sometimes as a single strand, enabling analysis even from very small amounts of
starting material. Known segments of DNA can easily be produced from a patient with a genetic
disease mutation. Modifications to the amplification technique can extract segments from a
completely unknown genome, or can generate just a single strand of an area of interest. It can
extract segments for insertion into a vector from a larger genome, which may be only available
in small quantities. Using a single set of 'vector primers', it can also analyze or extract fragments
that have already been inserted into vectors. Sequence-tagged sites is a process where PCR is
used as an indicator that a particular segment of a genome is present in a particular clone. In the
first chamber, the sample is heated to approximately 94 degrees Celsius with conducting wires
attached to the bottom of the chamber. These conducting wires are powered by resistors inside
of the device. Doors containing the sample within the chamber also contain the temperature.
Once the temperature inside the chamber reaches 94 degrees Celsius, the doors open and allow
32
the sample to channel into a separate cooling chamber. Here, a chemical reaction between
ammonium nitrate and water begins at the discretion of the program. This reaction is contained
within separate chambers within the cooling chamber, allowing the low temperature to be passed
to the sample without direct mixing of the solutions. Once the sample has cooled to 68 degrees
Celsius, doors open, pushing the sample into another heating chamber. This chamber uses the
same mechanism as the first heating chamber, however, the sample is only heated to 72 degrees
Celsius. This three step process is then repeated ten times to guarantee adequate DNA formation.
Each of the chambers is pictured in Figure 15, Figure 16, and Figure 17 in order of occurrence.
After completing PCR, the sample travels to the final step, spectrophotometry (Figure 18).
Researchers will look for DNA fragments that are only found in patients who have CF. These
DNA fragments are presented as ‘bands’ in the electrophoresis results. If the fragments are found
only in people who have the disease, it suggests that the fragments contain the DNA from a gene
variant that might mean a person is more susceptible to having the disease.
After the sample is pushed into the electrophoresis gel, a miniature spectrophotometer, placed
inside the NANOLYSER, will analyze (magnetic resonance spectroscopy-MRS resonance)
whether a cut segment in the gel electrophoresis matches up to the CFTR gene (189 kb in length,
with 27 exons and 26 introns, 1480 amino acids). If a match isn't or is made, a program either
turns on a green light to indicate that the user has cystic fibrosis or a red light to indicate the user
does not have cystic fibrosis respectively. A large scale view of the NANOLYSER is shown in
Figure 18.
33
7.4 NANOLYSER Program
As a result of the inclusion of PCR into our device, a computer program controlling a few of the
mechanical elements of our chip was necessary. This program, theoretically, would be a non-
disposable element that one could reuse for a variety of NANOLYSER devices. The chip would
control magnetic poles for electroosmotic flow, the doors of the various chambers, the
completion of a cooling reaction, and the light and spectrometer system for spectrophotometric
determination of cystic fibrosis.
Conducting plates located on either side of the channel before and after a PCR chamber alert the
program to open or close when the sample allows a current to be passed from one plate to the
other. Once the sample is in a chamber, the program allows the sample to be heated or cooled to
the desired temperature before the doors open once again. The program regulates the temperature
of the cooling chamber by only allowing portions of the ammonium nitrate and water solutions
to mix at any given time. In order to repeat the three PCR steps, the sample must be able to re-
cycle through them. The program makes this possible by switching the poles controlling
electroosmotic force within the device.
Finally, from the reading taken by the spectrometer, the program will illuminate a red or green
bulb located on the surface of the device indicating the presence or absence of cystic fibrosis
respectively.
7.5 Summary of Relevant Work and Research Topics in the Field
Prior research was found in order to back up the entirety of the NANOLYSER construction. The
goal was to make a design that easily diagnoses both people that have cystic fibrosis as well as
carriers. In order to do this, extensive research was conducted on cystic fibrosis and its
34
treatments. It was found that nationally, newborns are screened for cystic fibrosis using a genetic
test or a blood test. The genetic test shows whether a newborn has faulty CFTR genes and the
blood test shows whether a newborn's pancreas is working properly. In early childhood, a sweat
test is also conducted to confirm genetic test findings. This is also the most accurate test for
diagnosis is affected patients as it measure the amount of salt in sweat, which is done by
triggering sweating using chemicals on the arm and then using an electrode to run a mild
electrical current ("How Is Cystic Fibrosis Diagnosed?"). This is accurate due to the fact that
cystic fibrosis produce abnormal amounts of sodium and chloride across an epithelium.
The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked
glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered
in CF chromosomes and in residues believed to play a role in its function (Seng). Cystic fibrosis
is a result of a defective gene and its protein product (cystic fibrosis transmembrane conductance
regulator) which causes the body to produce unusually thick, sticky mucus. This mucus clogs the
lungs and leads to life-threatening lung infections and obstructs the pancreas and stops natural
enzymes from helping the body break down and absorb food.
8 NANOLYSER Strategy Development, Brainstorming, and Preliminary Design
8.1 NANOLYSER Strategy Development
One of the biggest challenges with the NANOLYSER project is that despite coming up with the
theory behind a NANOLYSER, our research group will not be able to manufacture an actual
working device due to outside restrictions and limitations. Instead, the team has to be able to
defend the integrity of the design using creativity and actual evidence from prior research
studies. From the beginning, team X6 wanted to design a chip that was straightforward and easy
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to understand, in order to provide an easy interface for the general user to handle, as well as for
ease of future client understanding. This way team X6 would not only be able to understand the
full potential of the device that was created, but when explaining the device and its theory to
others, the fundamentals wouldn’t get lost with complicated details and would keep things clear.
Another preference for the team was to make these chips easy to manufacture in order to
distribute to a larger number of people effortlessly. In the design process, the team decided to
keep as many features of the chip on the same plane as possible. It was thought that this design
philosophy would make the chips easier to make, if they were to actually be mass produced.
8.2 Design Parameters
8.2.1 Designs Considerations and Philosophy
The purpose of the NANOLYSER device was to create a device that could diagnose a disease
without the use of an entire lab. The design of the NANOLYSER was designed around this goal
as well as making a portable device and cost effective device. The design of the NANOLYSER
also was shaped around making the device user friendly and easy to read the diagnostics of the
device. The NANOLYSER utilizes several processes such as PCR, gel electrophoresis, and
spectrophotometry to diagnose a person with cystic fibrosis. The NANOLYSER device was
designed to be more cost effective than traditional diagnostics of cystic fibrosis which can
require an expensive full genetic test.
8.2.2 Fabrication Considerations
In order to create the NANOLYSER with a holistic approach, the materials used for fabrication
were analyzed and chosen to create the best functioning device for cystic fibrosis detection. The
chip itself was chosen to be fabricated out of silicon dioxide for two reasons. The first reason
being that chemical etching, the method used to create the features within the chip (filtering
36
pillars and mixing barriers), is most easily performed on silicon dioxide. The second, that silicon
dioxide creates an environment extremely conducive to creating electroosmotic flow. This is
because the silica donates a proton into the solution which creates a net positively charged
solution. The positively charged solution can be manipulated using electric fields created by
electrodes.
Gold nanowires were chosen to act as the conducting wires used to heat the sample for PCR.
Gold nanowires are both flexible and great conductors of electricity, two extremely influential
characteristics in the even heating of our sample in PCR. Similarly, the cooling step of PCR
required a reaction between ammonium nitrate and water. This reaction is commonly found in
ice-packs and was chosen for the reactants’ availability and ease of use.
8.2.3 Biological and Biochemical Considerations
The NANOLYSER that group X6 developed will be a single use diagnostic device for a genetic
disorder. The endonucleases and 10X CST buffer cannot be reused in the same reaction after its
initial use, and must be replenished after every use. The device itself would be disposed of whilst
the internal microSD programmed card would be interchanged between various NANOLYSER
shells. This would be more effective due to the fact that when trying to analyze tiny samples of
blood, the data must be as clean as possible in order to avoid misdiagnosis.
In order to take into account the excess waste coming from disposing each external shell after
every use, new methods of figuring out how to recycle these parts will have to be developed.
Despite this, having the NANOLYSER stay single use will outweigh these challenges by
reducing contamination through overuse of the microneedle and maintaining high quality of data
analysis for diagnosis.
37
As mentioned earlier, the used device will be recycle to maintain environmental friendliness.
This will enable all of the parts to potentially be reused and cut down on the effect
manufacturing the devices has on the environment.
Team X6's NANOLYSER will be extremely user-friendly for both hospital and personal use, as
the only user input for the device that is needed is a small sample of blood collected by the user
pricking his or her finger on the microneedle. Microneedles cause nominal bleeding once the
necessary amount of blood is drawn, allowing for maximum comfort for the patient.
9 NANOLYSER Final Design
The design of our NANOLYSER went through a variety of steps before attaining its final form.
The features included on the NANOLYSER follow a series of steps designed to break down the
blood sample in order to separate an analyte on which adequate disease determination can be
conducted. The device contains separation, mixing, heating, cooling, and determination steps,
each with its own unique process.
9.1 Explanation of Final Design
9.1.1 How It Works
The sample can be obtained by pricking the subject with a hypodermic needle, with the person’s
blood pressure funneling the sample into the device. Electro-osmotic force is then used to move
the sample through the chip and through the device. Micro-sized pillars will then separate the
blood plasma from the cells, allowing the white blood cells to pass through while keeping back
the red blood cells. The micro-sized pillars are 0.08 mm in diameter and are 0.088 mm away
from each other. The plasma and white blood cells continue through the channel until the
mixture reaches some wedge barriers that mix the sample by disrupting flow.
38
The sample will then be mixed with suitable reagents before subjecting the solution to
polymerase chain reaction (PCR), using heating and cooling chambers. PCR is used to create
copies of a selected part of a DNA strand and isolate it. The new solution is then moved into a
heating chamber to begin PCR where a conductive switch will be triggered that stops electro-
osmotic flow and shuts the chambers doors. The heating chamber will heat the sample to
approximately 94 degrees celsius (ºC) using resistors to denature the sample. After the sample is
in the heating chamber for appropriate amount of time, the electro-osmotic force is turned back
on and the heating chamber doors are opened. The sample then enter a cooling chamber and a
conductive switch is activated that stops the electro-osmotic force and closes the cooling
chambers doors. The cooling chamber will cool the sample down to 40 ºC using an endothermic
chemical reaction to anneal the sample. After the sample is cooled in the cooling chamber for an
appropriate amount of time, the electro-osmotic force is restored and the cooling chamber doors
are opened. The sample then enters another heating chamber where a conductive switch is
activated that stops the electro-osmotic force and shuts the doors of the heating chamber. The
heating chamber then heats the sample to 72 ºC using resistors. After the sample heats for an
appropriate amount of time, the electro-osmotic force is restored and the heating chamber doors
open. The electro-osmotic force is then put into reverse and the sample goes back to the first
heating chamber and this process is repeated ten times. After the tenth time of heating the sample
to 72 ºC, the sample then moves through a gel and gel electrophoresis is performed. A light
sensors then is able to detect if the protein is present. If the protein is present then the green light
will turn on while if the protein is not present the red light will turn on.
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9.1.2 Features
The design of our NANOLYSER went through a variety of steps before attaining its final form.
The features included on the NANOLYSER follow a series of steps designed to break down the
blood sample in order to separate an analyte on which adequate disease determination can be
conducted. The device contains separation, mixing, heating, cooling, and determination steps,
each with its own unique process.
9.2 Advantages of Design
Some advantages of the NANOLYSER device include its portability and readability. The
NANOLYSER device is small and can be transported unlike a research lab. The device also
gives back a green light if the user has cystic fibrosis and a red light if the user does not have
cystic fibrosis. The device is also able to diagnose a carrier of cystic fibrosis as well as people
who have the disease. The device is cheaper compared to a full genetic testing that would be
required to determine if a person was a carrier of cystic fibrosis. The device can also make a
diagnosis of a cystic fibrosis faster than a lab can diagnosis the disease. The device is relatively
painless because it utilizes microneedles and the device also requires less blood than full genetic
testing requires.
9.3 Disadvantages of Design
While the NANOLYSER design would have many advantages if developed, it would also have
its disadvantages. These disadvantages manifest themselves in both the cost effectiveness and the
functionality of the device.
First, the current design of the device implements the use of gold nanowires as conducting wires
for the two heating stages of PCR. From estimates gathered from sigmaaldrich.com, 10 mL of
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gold nanowires cost approximately $411. This results in a device that is much more expensive
than initially intended. This problem could be solved through research into conducting wires
made of other, less expensive materials, possibly phosphorous infused silicon.
Secondly, in order to function properly, the device must be completely still once the device is
prepared for use. This preparation specifically refers to the injection of the lysing agent,
10XCST, and the agarose electrophoresis gel into the NANOLYSER. Because the lysing agent
and gel are not a part of the device initially, they must be manually put in their respective places
within the channel. Thus, they are not contained or secured by anything other than the
intermolecular forces that hold them close. If the chip is tilted, these components may move out
of position, resulting in a malfunctioning NANOLYSER. To combat this, the white blood cells
could be lysed before moved into the NANOLYSER and an alternative CTFR gene detection
device could be used. These solutions, however, would result in significant further research and
changes to the device itself.
10 NANOLYSER Summary and Conclusions
10.1 Summaries
10.1.1 Design
This aspect of research called for the theoretical development of a NANOLYSER device capable
of making diagnosis of a disease from a blood sample. It was pertinent to implement logical and
sensible nanotechnology processes so that the device is as real as possible. While the device
itself may have been theoretical, the processes contained within the chip are backed by real
world data and a structural make-up of the device was created in SolidWorks. The purpose of
including real-world science into our device was to gain experience analyzing the components of
41
medical diagnostics and of nanotechnology processes. The complete NANOLYSER device is
shown in Figure 19.
10.1.2 Practicality of Chip Design
The chip design should be practical for actual diagnosis of cystic fibrosis. The NANOLYSER
design would be relatively expensive to actually produce, but there is a need for more
comfortable genetic disorder diagnosis. The created device would allow for patients to quickly
and easily detect if their blood seems to have a mutated CFTR gene. If the patient does have
these mutated genes, further testing could be performed to confirm or disconfirm final diagnosis.
Final diagnosis for cystic fibrosis is only possible through conducting a sweat test, or a full
genomic workup. Therefore this chip would allow for patients who may suspect cystic fibrosis,
or are potential carriers to use this at home treatment or for doctors who suspect a cystic fibrosis
patient to distribute for initial testing. The device would provide for a more comfortable and
expedient diagnosing time. Overall the practicality of the design seems to be high and that this
device could likely been seen in future medical applications, with further testing and research.
10.2 Conclusions
10.2.1 Results in Terms of Purpose
The group’s device aims to detect the presence of cystic fibrosis, or to determine if one is a
carrier for the genetic disorder. There is currently no way to detect cystic fibrosis in carriers
unless an entire genomic workup is conducted. Predicting early diagnosis can allow for inhibited
progression without treatment. The current methods of detection involve a sweat test where a
chemical is applied to the forearm and then a measurement of the chloride concentration level is
taken, and genetic testing. This NANOLYSER could potentially become a cheap and effective
method of early diagnosis of cystic fibrosis. By running a single drop of blood through this
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device incorporating nanotechnology with the use of a microneedle, methods for diagnoses
become simpler, cheaper, and more effective. By incorporating various methods that would
otherwise require a lab full of equipment and technicians, the space required for testing is
reduced to just a chip. The device also allows for a less invasive and less painful way of
diagnosing the presence of cystic fibrosis. This will allow for more individuals to be more
proactive in checking for cystic fibrosis as it will be cheaper and less painful this earlier
diagnosis would lead to the potential treatment or cure for cystic fibrosis in patients all over the
world.
10.2.2 Roadmap for Future Work
While our NANOLYSER device, theoretically, will be able to accurately diagnose cystic fibrosis
from a single blood sample, future research into an improved NANOLYSER device should be
done. Such research manifests itself in the use of more cost-efficient components. For example,
the possible implementation of phosphorus infused silica nanowires instead of gold nanowires
would reduce the cost of manufacturing a single chip drastically for 10mL of gold nanowires
costs $411.50. Additionally, significant progress could also be made in becoming independent
from the microchip program that controls the autonomous mechanical aspect of the chip. That is,
investigating natural physical and chemical processes that function similarly. Each of these
advancements, if made, would make the device more cost efficient and more practical as a
disposable device, all while maintaining its effectiveness as a cystic fibrosis diagnostic device.
43
11 Bibliography
Andersson, Helene. "Microfluidic Devices for Cellomics: A Review." Sensors and Actuators B:
Chemical 92.3 (2003): 315-25. Sciencedirect. Web. 26 Mar. 2014.
<http://www.sciencedirect.com/science/article/pii/S0925400503002661>.
"How Is Cystic Fibrosis Diagnosed?" - NHLBI, NIH. N.p., n.d. Web. 20 Apr. 2014.
Jiang, Hai. "Microfluidic Whole-blood Immunoassays." Microfluidic Nanofluid (2011): 941-64.
Springer Link. Web. 25 Mar. 2014.
<https://carmen.osu.edu/d2l/le/content/11289035/viewContent/6552918/View>.
Mercier-Bonin, M. "Journal of Colloid and Interface Science." Study of Bioadhesion on a flat
Plate with a Yeast/glass Model System (2004): 342-50. Web. 25 Mar. 2014.
<https://carmen.osu.edu/d2l/le/content/11289035/viewContent/6595482/View>.
“Reynolds number.” Encyclopedia Britannica. Web. 25 Mar. 2014.
Lab 2 Memo Write Up. 2043, January 10. www.carmen.osu.edu.
Nanofluidics Teaching Module. 2014, January 10. www.carmen.osu.edu.
1282.02–Lab 2: Microfluidics Procedure. 2014, January 10. www.carmen.osu.edu.
Seng H. Cheng, Richard J. Gregory, John Marshall, Sucharita Paul, David W. Souza, Gary A.
White, Catherine R. O'Riordan, Alan E. Smith, Defective intracellular transport and
processing of CFTR is the molecular basis of most cystic fibrosis, Cell, Volume 63, Issue
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4, 16 November 1990, Pages 827-834, ISSN 0092-8674, http://dx.doi.org/10.1016/0092-
8674(90)90148-8. (http://www.sciencedirect.com/science/article/pii/0092867490901488)
45
Appendix A
Experimental Procedure
The experiment should follow the step by step procedure below.
1. First, obtain two 2 inch petri dishes, a stirring rod, a cup, latex gloves, a vacuum pump,
desiccator, syringes, PDMS resin, and curing agent.
2. Place the acrylic chip, provided by a lab instructor, in a petri dish with the patterned side up.
3. We will make PDMS by mixing a base and a curing agent in a 10 to 1.05 ratio.
4. Make sure a total of 15.9 g of PDMS will be made.
5. Calculate the volume needed to get 15.9 g of PDMS based on the specific gravity of the
substance. The volume of the base should be 13.97 mL and the volume of curing agent
should be 1.47 mL.
6. Fill the 20 mL syringe with the base to 13.97 mL and the 3 mL syringe with the curing agent
to 1.47 mL.
7. Add the appropriate amount of each substance to the cup and make sure that they are
thoroughly mixed.
8. Remove the lids from the petri dishes.
9. Pour about half of the PDMS into the petri dish with the chip mold.
10. Then, pour the rest of the PDMS into the other petri dish.
11. There will likely be air bubbles in the petri dishes. Use the vacuum pump and desiccator to
get as many air bubbles out as possible.
12. Once there are no air bubbles (or at least very few), disconnect the vacuum and desiccator
and replace the wire mesh in the desiccator.
13. Replace the lids on the petri dishes.
14. Put the petri dishes in an oven at around 95 ºF overnight to cure.
ii
15. Clean the stirring rod well with paper towels.
16. Wipe up any spilled PDMS.
17. Once the PDMS is done curing, carefully demold the chip out of the petri dish. Then, weigh
a beaker of a triple beam balance.
18. Once the mass of the beaker is known, adjust the balance so that it reads 2 g more than the
beakers mass.
19. Add yeast to the beaker until the balance zero out again.
20. Add 10 mL of deionized water to the beaker.
21. Stir the contents of the beaker to dissolve the yeast.
22. Wait 20 minute of the yeast to incubate in the water.
23. Stir the water slightly and then take about 5 mL of the solution in a syringe.
24. Insert the tip of the syringe into the entrance of the chip.
25. Slowly pump the yeast water through the chip until it just barely comes out of the exit. Take
care as to not force the layers of the chip apart, or to deform the sides of the channels.
26. The chip should sit for exactly 20 minutes to give time for the yeast cells to adhere to the
channels.
27. As the chip sits for 20 minutes, the microfluidic system should be set up which includes the
stand, the tygon tubes, and the syringe. The tubes should be filled completely with water by
submerging each of them in water and pumping water through them until no bubbles of air
come out. The 20 mL syringe should be placed at the height of the 20 cm from the height of
the outlet tube. The inlet tube should be connected to the 20 mL syringe and the outlet tube
and the inlet tube should be positioned for the experiment. Both the inlet and outlet tubes
should be clamped.
iii
28. Make sure the chip is completely dried before starting the experiment using Kim wipes.
29. Place the plastic adaptor on the longer inlet tube into the inlet of the channel with yeast cells.
30. Place the plastic adaptor on the shorter outlet tube into the outlet of the channel with yeast
cells.
31. Place the cub under the free end of the outlet tube to gather water.
32. Fill the syringe with water to the top
33. Remove the clamps on the inlet and outlet tubes and allow flow through the chip for exactly
5 minutes at low pressure flush. Reclamp both the inlet and outlet tubes after 5 minutes to
prevent anymore flow.
34. After the initial flush, look at the channels under the microscope to make sure that there are
no cells still flowing and that there are no clumps of yeast cells at the entrance port. If there
are still cells flowing or there are clumps of cells at the entrance port than an additional 2
minute flush should be performed.
35. Continue 2 minute flush’s until the there are no clumps of yeast cells at the entrance and
there are no cells that are still flowing
36. The first height of the syringe after the flush should be 35 cm and the height should be
recorded.
37. Weigh the empty dry cup using a balance and record the mass of the cup.
38. The water level in the syringe should always be filled to the top.
39. The part of the channel that will be observed should be marked using a dry erase marker.
40. Look at the part of the channel that was marked under the microscope at the lowest
magnification and take a picture and count the number of yeast cells.
41. Attach the chip to the inlet and outlet tubes.
iv
42. Unclamp the tubes and start the timer.
43. Allow flow for exactly 2 minutes and then reclamp the inlet and outlet tubes.
44. View the channel under the microscope and take a picture and observe the changes in the
number of yeast cells. Record the number of yeast cells
45. Weigh cup with the water that came out of the outlet tube during the 2 minutes
46. Refill the syringe to the top with water.
47. Repeat steps 36-46 at increasing heights until 50 % of the initial cells after the flush have
been remove.
48. Clean the channels by sonicating the chip and making sure all yeast cells are gone using a
microscope.
49. At least 10 data points should be collected - 5 for the patterned and 5 for the unpatterned.
v
Figures
i
Figure 1: Isometric View of the Channel Mesh Used In Flow Simulation.
Figure 2: Goals Plot Indicating Convergence of Maximum and Average Velocity for
a Pressure Difference of 49,000 dyne/cm2.
ii
Figure 3: Cross Sectional Velocity Contour for a Pressure Difference of 49,000
dyne/cm2.
Figure 4: Shear Stress Contour for a Pressure Difference of 49,000 dyne/cm2.
iii
Figure 5: Microfluidics Program
clear;clc;disp('Ryan Schoell')disp('Engineering 1282.02H')disp('PAC 10:20')fprintf('This program generates average velocity, shear stress at the wall,\n Reynolds, enterance length, and a missing parameter based on\n volumetric flow rate, width, height, length, delta P, and viscosity.\n This program also generates a plot of velcoity vs. thickness \n and shear stress vs. thickness.\n\n')%Ask the user to input a zero for the missing parameterdisp('Please put a value of zero for the parameter not given')%Use inputs to have the user input the information for the parametersfor k=1:4Q=input('Please input a volumetric flow rate in cm^3/s: ');W=input('Please input a width in cm: ');H=input('Please input a height in cm: ');L=input('Please input a length in cm: ');DP=input('Please input a delta P in dynes/cm^2: ');U1=input('Please input a viscosity in cp: ');%Change the units of viscosity from cp to g/cm-sU=U1/100;%Use if statements to solve for the missing parameterif Q==0Q=(DP*H^3*W)/(12*U*L);elseif W==0W=(12*Q*U*L)/(DP*H^3);elseif H==0H=(12*Q*U*L)/(DP*W);elseif L==0L=(DP*H^3*W)/(12*U*Q);elseif DP==0DP=(12*Q*U*L)/(H^3*W);elseU=(DP*H^3*W)/(12*L*Q);end%Solve for Average velocity, shear stress at the wall, reynolds number, and%enterance lengthAV=(DP*H^2)/(12*U*L);T=DP*H/L/2;Re=AV*((4*W*H)/(2*W+2*H)/U);Le=0.06*Re*(4*W*H)/(2*H+2*W);%Use fprintf to display the results in an organized fashionfprintf('Volumetric Flow Rate Width Height Length')fprintf('\n %3.3f cm^3/s %3.3f cm %3.3f cm %3.3f cm',Q,W,H,L)
iv
fprintf('\n\nDelta P Viscosity Average Velocity')fprintf('\n%3.3f dyne/cm^2 %3.3f g/cm-s %3f cm/s',DP,U,AV)fprintf('\n\nShear Stress at wall Reynolds Number Enterance Length')fprintf('\n%3.10f dynes/cm^2 %3.3f %3.3f cm\n',T,Re,Le)endUse a vector to express the point in the channel thicknessy=[-H/2:0.01:H/2];%Solve for velocity based on point in the channel thicknessV=(DP.*H.^2)./(8.*U.*L).*(1-4.*y.^2./H.^2);%Solve for shear stress based on a point in the channel thicknessT1=DP./L.*y;figure(1)%Plot velocity vs. thicknessplot(y,V)%Label the graphtitle('Velocity across the Channel')xlabel('Thickness (cm)')ylabel('Velocity (cm/s)')figure(2)%Plot shear stress vs. thicknessplot(y,T1)%Label the graphtitle('Shear Stress acoss the Channel')xlabel('Thickness (cm)')ylabel('Shear Stress (dyne/cm^2)')
v
Figure 6: Graph of Theoretical Flow Rate versus Water Height.
Figure 7: Graph of Theoretical Pressure versus Water Height.
vi
Figure 8: Graph of Theoretical Flow Rate versus Theoretical Pressure.
vii
Figure 9: Chip and Chip Holder Assembly
viii
Figure 10: Shear stress from height versus yeast cell count.
ix
Figure 11: Shear stress from flow rate versus yeast cell count.
x
Figure 12: Microneedle process.
xi
Figure 13: Filtering step of the NANOLYSER device.
Figure 14: Mixing stage of the NANOLYSER device.
xii
Figure 15: First heating stage of PCR.
Figure 16: Cooling stage of PCR.
xiii
Figure 17: Second heating stage of PCR.
Figure 18: Spectrophotometry stage of the NANOLYSER device.
Figure 19: Complete NANOLYSER device.
xiv
Figure 20: Yeast cell adhesion in a microfluidics channel.
xv
Tables
Table 1: Mesh details for the flow simulation
Mesh Details
Number of Cells - X 40
Number of Cells - Y 40
Number of Cells - Z 80
Type of Cells Water
Cell Shape Spheres
Number of Pressure Inlets 1
Number of Pressure Outlets 1
Number of Fluid Cells 70626
Number of Partial Cells 43550
Number of Refinements 0
Number of Cells After Refinement 40, 40, 80
Dimension of Mesh .006 m x .003 m x .028 m
i
Table 2: Simulation conditions for a pressure difference of 49,000 dyne/cm2
Simulation Conditions - High Pressure
Inlet Pressure 106225 Pa
Outlet Pressure 101325 Pa
Density 998.2 kg/m^3
Viscosity .001 Pa-s
Number of Iterations 80
Number of Iterations per Travel 129
Delta Value 3.99e-7 m/s
Maximum Velocity 0.315717 m/s
Wall shear Stress 10.66 Pa
ii
Table 3: Simulation conditions for a pressure difference of 39,200 dyne/cm2
Simulation Conditions - Medium Pressure
Inlet Pressure 105245 Pa
Outlet Pressure 101325 Pa
Density 998.2 kg/m^3
Viscosity .001 Pa-s
Number of Iterations 80
Number of Iterations per Travel 129
Delta Value 9.56e-7 m/s
Maximum Velocity 0.252613 m/s
Wall shear Stress 8.52 Pa
iii
Table 4: Simulation conditions for a pressure difference of 29,400 dyne/cm2
Simulation Conditions - Low Pressure
Inlet Pressure 104265 Pa
Outlet Pressure 101325 Pa
Density 998.2 kg/m^3
Viscosity .001 Pa-s
Number of Iterations 80
Number of Iterations per Travel 129
Delta Value 1.10E-06
Maximum Velocity 0.189514 m/s
Wall shear Stress 6.39 Pa
iv
Table 5: Experimental and theoretical values
Height (m)
Length (m)
Pressure Drop (kg/m-s2)
Reynolds Number
Data / Calculation Type
21 22 33 Mean
0.508 0.0172 109.53136 0.0086962 time (s) 120.00 120.00 120.00 120.00
mass (kg) 0.0003 0.0003 0.0003 0.0003
Volume (m3): 0.0000 0.0000 0.0000 3.01E-07
Q (m3/s) 0.0000 0.0000 0.0000 2.50E-09
Vavg(m/s) 0.0342 0.0342 0.0342 0.0342
v
Table 6: Relationship between Shear Stresses and Number of Yeast Cells
Height (cm)
Volume (mL)
Time (min)
# of cells after flow
Flow Rate (mL/s)
Pressure (dyne/cm2) from flow rate
Shear Stress (dyne/cm2) from flow rate
Pressure (dyne/cm2) from height
Shear Stress (dyne/cm2) from height
Data Point 1
Height 1 (Low Pressure Flush) 25.4 6.46 3 675 0.035888 4.486 0.0179444 24892 99.568Height 2 30 13.91 2 273 0.11599 14.499 0.057995 29400 117.6Height 3 40 22.2 2 187 0.185 23.125 0.0925 39200 156.8Height 4 50 31.52 2 180 0.262666 32.833 0.131333 49000 196
Data Point 2
Height 1 (Low Pressure Flush) 25.4 5.45 3 276 0.030277 3.785 0.0151385 24892 99.568Height 2 30 13.42 2 95 0.11183 13.979 0.055915 29400 117.6Height 3 40 21.58 2 33 0.17983 22.479 0.089915 39200 156.8Height 4 50 31.27 2 23 0.26058 32.572 0.13029 49000 196
Data Point 3
Height 1 (Low Pressure Flush) 25.4 6.93 3 215 0.0385 4.812 0.01925 24892 99.568Height 2 30 14.97 2 121 0.12475 15.594 0.062375 29400 117.6Height 3 40 23.04 2 63 0.192 24 0.096 39200 156.8Height 4 50 32.76 2 27 0.273 34.125 0.1365 49000 196
Average Height 1 25.4 N/A 3 388.666667 0.03488833 4.361 0.0174443 24892 99.568Height 2 30 N/A 2 163 0.11752333 14.69066667 0.058761667 29400 117.6Height 3 40 N/A 2 94.3333333 0.18561 23.20133333 0.092805 39200 156.8Height 4 50 N/A 2 76.6666667 0.26541533 33.17666667 0.132707667 49000 196
Percent Difference Point 1 Height 1 25.4 6.46 3 -74% -3% -3% -3% 0% 0%
Height 2 30 13.91 2 -67% 1% 1% 1% 0% 0%Height 3 40 22.2 2 -98% 0% 0% 0% 0% 0%Height 4 50 31.52 2 -135% 1% 1% 1% 0% 0%
Percent Difference Point 2 Height 1 25.4 5.45 3 29% 13% 13% 13% 0% 0%
Height 2 30 13.42 2 42% 5% 5% 5% 0% 0%
i
Height (cm)
Volume (mL)
Time (min)
# of cells after flow
Flow Rate (mL/s)
Pressure (dyne/cm2) from flow rate
Shear Stress (dyne/cm2) from flow rate
Pressure (dyne/cm2) from height
Shear Stress (dyne/cm2) from height
Height 3 40 21.58 2 65% 3% 3% 3% 0% 0%Height 4 50 31.27 2 70% 2% 2% 2% 0% 0%
Percent Difference Point 3 Height 1 25.4 6.93 3 45% -10% -10% -10% 0% 0%
Height 2 30 14.97 2 26% -6% -6% -6% 0% 0%Height 3 40 23.04 2 33% -3% -3% -3% 0% 0%Height 4 50 32.76 2 65% -3% -3% -3% 0% 0%
ii
Table 7: Microfluidics statistical results
Significance Level α: 0.05
α/2: 0.025
Degrees of Freedom: 7
P-value: 0.97
i