Microfluidic Blood Separation for Plasma Testing

Jeremy Hartman, Charles Linskey, Kim Terrizzi, Deborah Pano, Carmen Wu

December 18, 2015

Submitted to:
NORTHEASTERN UNIVERSITY
Department of Chemical Engineering
CHME 2311 Transport 1 Laboratory
Dr. Landherr

B.1

Table of Contents
Table of Contents ...

Table of Figures ...

iii

Table of Tables

iv

Executive Summary ...............

1. Introduction .

2. Background ....

2.1 Plasma .

2.2 Bent Microchannels.

2.3 Capillary Filtration Through Microbeads

2.4 Comparison of Methods.......

3. Experimental Method .....

3.1 Preparation...

3.2 In-Laboratory Execution..

3.3 Expected Results..

10

3.4 Safety Analysis..

11

4. Timeline...

12

5. Cost Analysis

15

6. Broader Impacts ..

18

7. References

19

Appendix A: Experimental Procedure..

A.1

Appendix B: Raw Data Table.

B.1

Appendix C: Sample Calculations.

C.1

Table of Figures
Figure 1 Bent Microchannel Diagram...............

Figure 2 Microbead Filtration Diagram................

Figure 3 Design of Bead Packed and Bent Channel Chips.

Figure 4 Experimental Timeline.

14

iii

Table of Tables
Table 1: Cost Analysis...............

15

Table A-1: Data Table Template for Raw Data Collected...

B.1

iv

Executive Summary
In response to the challenge of engineering better medicines posed by the National Academy
of Engineering, two microfluidics-based separation methods used to extract plasma from whole
blood will be experimentally evaluated to determine the better method. Optimally, the better method
will be able to operate at a higher flowrate without compromising purity of separated components.
Researchers can obtain a great amount of information about different types of cancer and other
diseases by collecting samples of cells and proteins from the patients bloodstream. Microfluidics can
isolate blood components on small chips based on various factors including differing geometry, fluid
forces, and external force fields with minimal cell damage.
The first method to be tested separates blood components via a bent microchannel, which
results in a centrifugal force. The centrifugal force that is induced during this microscale process is
minimal and is exerted over a short period of time, preventing cell lysis. As blood flows through this
bent microchannel, a fork with two microchannels of varying diameters forces the less dense plasma
to be separated into the smaller microchannel away from the blood cells. For this method, channel
size and flowrate of the blood pump will be varied to determine the effect on the flowrate of
separated plasma. One design will have a 100 um main channel with a 20 um side channel and the
second design will have a 100 um main channel with a 40 um side channel. Each of these chips will
be tested at three different pump flowrates: 0.5 uL/s, one uL/s, and two uL/s.
The second method separates blood components via microbeads that act as a filter. The use of
microbeads circumvents common problems associated with conventional filtration techniques such
as clogging or cell lysis. The beads are used to block a microchannel and capillary action draws the
blood through the beads. As the blood passes through the beads, the blood cells cannot pass as easily
because of their size and are separated from the plasma, and the plasma is pulled into the
microchannel. The bead size will be kept constant. The size of the microchannel will be varied to
determine its effect on flowrate for this method. The first plate design will have a 100 um opening to
the capillary and the second plate design will have a 75 um opening.
Flowrate will be calculated based on the time needed to collect five microliters of plasma. A
preliminary purity analysis will be performed on this collected plasma via spectrophotometry to
determine whether there is any contamination from blood cells. Results from the two methods will be
compared to determine the more time-efficient method. Theoretically, the bent microchannel method
should give better yield time when compared to the bead packed channel, but the plasma may not be
as pure. The purity may vary greatly with the bent microchannel, as the applied flowrate will have a
direct impact on the plasma filtered off.
The budget allotted for this experiment is $20,000 and the time allotted is 3.5 hours. The bulk
of the budget will be used to purchase a spectrophotometer (cost: $7620.73) that is able to measure
the transmittance for ultra-micro amounts. The total amount that will be spent is estimated to be
$14,152.82, which falls well within the budget. The experiment should take 145 minutes to complete.
The majority of the work will be preparation, which includes machining molds and constructing
chips through multilayer soft lithography. This preparation will be outsourced to Stanford
Microfluidics Foundry, and the completed chips are expected to arrive within three weeks.
This experiment will contribute to developing a more effective method to separate plasma
quickly and efficiently from blood cells to allow medical testing to be conducted more rapidly. In the
future, continued experiments on microfluidic blood separation will refine and improve the process
until it can be effectively implemented in the medical industry.
1

1. Introduction
Engineering Better Medicines has been identified as one of the Grand Challenges for
Engineering published by the National Academy of Engineering . This challenge generally
[1]

covers projects engineers are currently working on relating to developing new systems for using
genetic information, detecting minute changes in the body, assessing new drugs, and delivering
vaccines .
[1]

Many research and development projects currently focus on every aspect of medicine;
there are approximately 250 medicines currently in development that specifically target
leukemia, lymphoma, and other cancers of the blood . These blood cancers account for nearly
[2]

ten percent of all cancer diagnoses in the United States, and approximately ten percent of cancer
patients succumb to the diseases each year . The most promising treatments currently in
[3]

development for these cancers relate to an improved understanding of bone marrow cells and
how the genes in their DNA can cause the disease. A greater understanding in the genetic cause
can allow for more effective treatments to be developed and tailored to a patients individual
needs .
[4]

Clinicians can learn a vast amount about different types of cancer and other diseases by
collecting samples of cells and proteins from the patients blood stream. To analyze these cells
and proteins, blood must be separated into different components. One such method for harvesting
these components is through microfluidic separation and analysis . Based on various factors
[5]

including differing geometry, fluid forces, and external force fields, different parts of a patients
blood can be separated and isolated on small chips. Separation of the blood components allows
for the targeted cells and proteins to be isolated and removed for testing.
A majority of blood tests require that the whole blood taken from the patient be separated
from the red and white blood cells leaving just the plasma behind. This process, called blood
fractionation, allows for the most efficient use of the blood sample; different parts of the blood
are used for different tests, and separating the necessary components allows for the most accurate
results with minimal contamination . Most commonly, blood is separated through centrifugation,
[6]

in which the blood is placed in a vial and spun through a cycle. During this cycle, the different
2

components of the blood separate based on differing densities. Unfortunately, the centrifuging
process requires more time and, under the constant centrifugal forces, red and white blood cells
can lyse, resulting in contamination of the final sample . In an attempt to improve this process,
[7]

microfluidics are being employed to separate the blood in less time than centrifuging, and also
with less contamination of the collected plasma.
This investigation will focus on two different methods that are currently being tested and
have been proven to be effective at a small scale for microfluidic blood separation. The first
method consists of using a bent microchannel to separate the blood cells from the plasma. The
second method consists of capillary action pulling the plasma through a series of beads and up a
microchannel while the blood cells are caught and left behind. These two methods will be
experimentally compared to see how they perform under a variety of pressure settings and a
variety of microchannel sizes. The final results will consist of the volumetric flowrate,
determined by measuring the time to collect five microliters of plasma, and the relative amount
of contamination in the plasma sample, measured by the light transmittance compared to a pure
plasma sample. The quantitative results from each trial can be compared to other individual
trials, and also to the initial experiments performed with these chip designs.
2. Background

Microfluidics refers to designing and manufacturing devices that can control and
manipulate flow of fluids in the microliter and nanoliter scales . Fluids operating at these
[8]

microscales behave rather differently when compared to the macro-scale behavior. Most
importantly, the Reynolds Number is much lower at the microscale when compared to the macro
scale, allowing flows to be highly laminar. The Reynolds Number is found using the formula

Eqn. 1
where is the density of the fluid, v is the velocity of the fluid, D is the diameter of the channel,
and is the dynamic viscosity of the fluid. Because the diameter of the microchannels is very
small, the Reynolds Number is proportionally small. The highly laminar characteristics of the
3

fluid flow allow for the development of a variety of novel applications, including blood plasma
separation.
2.1 Plasma
Plasma is the liquid portion of blood in which red blood cells, white blood cells and
platelets are suspended. It accounts for 55 percent of the volume of blood. The plasma mostly
contains water in which antibodies and proteins are suspended . Separating plasma from blood is
[9]

necessary to perform protein plasma screens. Total protein levels in the plasma can indicate a
number of health disorders. Abnormal protein levels can indicate hepatitis, human
immunodeficiency virus (HIV), leukemia, and other disorders .
[10]

For example, in the presence of inflammation, the body produces and releases extra
protein from the site of inflammation . Several tests can be run to detect the resulting increase in
[11]

protein. The first is testing erythrocyte sedimentation rate (ESR), which tests how quickly blood
cells naturally separate from the plasma with the aid of gravity. A high ESR is caused by certain
proteins covering red blood cells, which then stick to other red blood cells, and thus will fall
more quickly . A plasma viscosity (PV) test can also be conducted, and works in much the same
[11]

way; that is, the plasma is more viscous when the excess of proteins causes the red blood cells to
attach to one another. PV tests are more difficult to perform than ESR tests; therefore they are
less widely used . C-reactive protein (CRP) levels can also be specifically measured to
[11]

determine if a patient has an abnormal condition . Plasma can also be tested for specific
[11]

biomarkers that indicate particular diseases. For example, presence of microRNA in plasma can
serve as a biomarker of cancer .
[12]

2.2 Bent Microchannels

One promising separation method currently being developed involves utilizing bent
microchannels to facilitate the separation of blood cells and plasma. The whole blood, containing
both the blood cells and plasma, is pumped through a microchannel that experiences a sharp
bend. As the fluid travels through the bend, a centrifugal force is induced at the microscale and
the blood begins to separate based on the density of the components. This method employs the
same principle as traditional centrifugation, but the induced force is at a much smaller magnitude
and is applied for a much shorter time, resulting in fewer blood cells lysing. The red blood cells
are more dense than the plasma, resulting in the red blood cells moving to the outside of the bend
and the plasma moves towards the inside of the bend. As the partially separated blood comes out
of the bend, it reaches a fork where a second microchannel with a smaller diameter branches off.
4

The smaller channel is placed on the inside of the bend, allowing it to be closer to the lower
density fluid. Because the plasma, which is less dense, is travelling closer to this branch, it is
naturally skimmed off of the primary stream while the blood cells and some unseparated plasma
continue down the main channel. The separated plasma can then be collected and used for
whatever tests necessary . Figure 1 depicts the top view of a bent channel chip. The experiments
[13]

that have been completed by researchers showed positive results using a 100 micrometer primary
channel and a 20 micrometer plasma channel.

Figure 1: Bent Microchannel Diagram

2.3 Capillary Filtration through Microbeads
Another promising technique involves separation via filtration using microbeads.
Traditional filtration methods, such as running the fluid through a porous filter, are prone to
clogging or causing blood cells lysis. One solution to the filtration problems is using microbeads
instead of a traditional porous filter. The beads are placed in a reservoir and a negative pressure
is drawn to pull the beads against the opening of the microchannel. Once the beads are in place,
the vacuum is relieved and the beads are left resting at the entrance of the microchannel. The
whole blood is then placed in the reservoir with the beads and capillary action draws the blood
through the beads and into the channel. Capillary action is a result of the complex adhesive and
cohesive forces at play between the fluid and the surfaces it is in contact with, such as the beads
and the sides of the microchannel . As the whole blood passes through the beads, the plasma can
[14]

easily pass through the beads and into the microchannel, while the blood cells can not travel
through the gaps in the beads and are left behind. Once the plasma is pulled into the
microchannel, it will collect in a secondary reservoir until the final volume is collected. This
entire system operates as a result of capillary action so no external pressure is required. Figure 2
5

depicts a preliminary sketch of this configuration . This method has also been tested and shown
[16]

positive results for separating the plasma from very small volumes of blood.

Figure 2: Microbead Filtration Diagram

2.4 Comparison of Methods
This experiment is intended to compare the results of the previously described methods,
using altered conditions from the original experimental configurations. Microfluidics are highly
dependent on the dimensions of the microchannels and other parameters of the system, which
will be the primary focus. For the bent microchannels, two different size channels will be
utilized, with each channel tested under three different applied flow rates. Increasing the flow
rate of the whole human blood pumped into the system should increase the flow rate of the
separated plasma, but it is possible that the Reynolds Number may increase too much resulting in
more turbulent flow, which can contribute to lower purity in the collected plasma. Increasing the
diameter of the channel may also result in a higher Reynolds Number and more contaminated
plasma. For the microbead filtration, no external pressure is applied in any situation, but the size
of the microchannel will be altered compared to the previously completed experiment. As the
size of the microchannel increases, the rate of capillary movement should increase, however
there will be an eventual size when the tube is too wide to induce the movement. To compare the
efficacy of each method, the time to collect five microliters of plasma will be measured, allowing
for calculation of a flow rate, and the relative contamination of the sample will be measured
using a spectrophotometer. A sample of pure plasma will be used to determine the full
transmittance level, and then the spectrophotometer can measure if there is an optical
contamination in the collected samples as a result of the presence of blood cells. Using the data

collected, it will be possible to determine which process is more promising for efficient blood
separation based on the time to collect the sample as well as the purity of the final sample.
Using the absorbance data collected with the spectrophotometer, Beers Law can be used
to determine the concentration of red blood cells in the sample of plasma. Using the formula:
A=bc

Eqn. 2

A is the measured absorbance of the sample, is the molar absorptivity, b is the path length of
the sample, and c is the concentration in the sample. This formula can be used to quantify the
purity of the collected plasma based on the concentration of red blood cells in the sample.
Ideally, for accurate testing to be done on the plasma, the concentration of red blood cells should
be minimized to a maximum limit of five to ten percent .
[17]

3. Method for Comparison of Plasma Separation Techniques

3.1 Preparation
Four separate molds will be designed in AutoCAD based on the four different types of
plates needed: one mold for the bent-channel design with a 100um main channel and a 20um side
channel extending at 90 degrees from the bend, another of the bent-channel design with a 200um
main channel and 40um side channel extending at 90 degrees from the bend, a mold for the bead
packed channel design with a 100um opening to the capillary, and a mold for the bead packed
channel design with a 75um opening to the capillary. The differences will allow comparison
between the two methods of drawing liquid into the reservoir and the effect of different channel
sizes in both methods. Molds for chips will be created according to a procedure described in the
literature at the Stanford Microfluidics Foundry.
[14]

Figure 3: Design of Bead Packed Chips (left) and Bent Microchannel Chips (right)
7

At the foundry three chips will be created from each bead packed channel mold, and three
chips each will be created from the bent channel designs. These chips will be produces using
multilayer soft-lithography of Polydimethyl Siloxane (PDMS), a polymer that is compatible with
human blood and has minimal contamination risks for sample flowing through the microchannel.
All of these chips will then be sterilized and shipped to the laboratory for use. Multiple attempts
may be required to produce a functional chip from each mold of the creation of each chip, but
only fully functional chips will be shipped, this is factored into the expected cost and timeline.
The single-syringe pump will be calibrated for appropriate flowrates using the measurements of
the time required to pump out the known volume. Calibration of this equipment will ensure
accurate data for flowrate from the pump throughout the experiment.
All chips received from the Stanford Microfluidics Foundry will be tested to make sure
the channels are free of obstructions by running de-ionized water through the channels at a
known flow rate, using the calibrated single-syringe pump, then all chips will be dried. Although
this flow will not be comparable to the viscosity of the blood that will be flowing through it
during the experiment, it will be sufficient to ensure there are no obstructions in the capillaries.
In the chips made from the bead-packed mold designs, the opening of the channels will
be sealed with microbeads using the method described in a similar experiment.

[15]

First the

channel will be rinsed with DI water, then 100 micron diameter beads will be inserted and
pressure pulled through to block the channel opening. Phosphate Buffered Saline (PBS) will be
drawn through to seal these beads in place, then the 10 micron diameter beads will be drawn
through to block the channel on top of the larger beads and again sealed in place with PBS.

[16]

Capillaries in the tubes will be connected to reservoirs, which will be marked for a
volume of 5uL so that flowrate of plasma accumulation in the capillaries can be calculated from
the time required to fill reservoir up to 5uL.
The spectrophotometer will be calibrated for 100 percent transmittance by blanking the
equipment with a 5uL sample of pure plasma at a wavelength of 280nm , the wavelength used to
[19]

detect red blood cells. 5uL standards of 15 percent, 30 percent, 45 percent, 60 percent, 75 percent
and 100 percent isolated red blood cells in pure plasma will be made and measured on the
spectrophotometer. The data will be used to make a calibration curve, which will be used with
the measured spectrophotometer value to quantify the purity of the plasma.
3.2 In-Laboratory Execution
8

Chips printed from the molds will be attached to the bottom plate with clamps and the
syringe pump will be loaded with a syringe containing the desired amount of blood, then
attached through the opening into system on the bent channel chips. On the bent channel chips
80uL of whole human blood will be injected into the opening of the channel at the selected blood
flowrate using the syringe pump, and the timer will be started. One chip of each channel size will
be run at each of the following pump flowrates; 0.5uL/s, 1uL/s, and 2uL/s. The blood used will
contain five percent heparin in order to prevent clotting of blood inside the channels. On each
bent channel chip, this method will be repeated three times for each configuration, repeating the
same pressures and volume added on all of bent channels chips and washing the chip with 70
percent ethanol between each run.
On the bead packed channel chips 40uL of whole human blood will be injected into the
reservoir at the opening of the channel using a syringe and the timer will be started. The blood
used will contain 5 percent heparin in order to prevent clotting of blood inside the channels.
Flow on the bead packed chips will be monitored with proscopes, for a magnified view, until
plasma has accumulated in collection reservoir up to the mark for a volume of 5uL, then the
timer will be stopped. Time for each volume accumulation will be recorded in Table A-1. The
bead packed chips will be run concurrently for efficiency because of the length of time capillary
action takes to separate the plasma. Each design will be run under the same condition, adding the
blood and applying no pressure. Running three identical plates under the same condition will
result in each measurement being taken three times.
For all chip designs the flow will be monitored until plasma has accumulated in
collection reservoir up to the mark for a volume of 5uL, then the syringe pump will be shut off
and the timer will be stopped. Time and pump flowrate setting for each chip will be recorded in
Table A-1. Flowrate of plasma collected for all chips will be calculated based on time required to
fill the reservoir to 5uL and recorded for each chip in Table A-1.
After each run on a chip, of either design, the 5uL of plasma collected in the reservoir
will be collected with a syringe and transferred to a cuvette. Purity of these samples will be
tested in the cuvettes using the spectrophotometer. Spectrophotometer will be blanked with pure
plasma in an identical cuvette prior to running each sample, and readings will be recorded in
Table A-1 and compared to the calibration curve to quantify the purity of the sample collected
and whether any red blood cells are still present.

Concentrations will be calculated from the percent transmittance using Beers Law, and
recorded in Table A-1. Then concentrations of red blood cells will be used for comparison of the
purity of samples produced by different methods.
All cuvettes, chips, and other materials that will have contact with blood will be disposed
of in appropriately labeled biohazardous waste barrel to comply with appropriate safety
procedures. Syringes will be disposed of in appropriately labeled biohazardous sharps container
to comply with appropriate safety procedures.
3.3 Expected Results
The more effective method will likely be the bent channel filtration. The bead packed
microchannels method has been proven to be effective for very small volumes, but in practice it
may take too long to collect a clinically relevant quantity. The amount of plasma collected in this
experiment, 5 uL, is a sufficient amount for clinical use, and if the bead packed channels are not
able to collect this much, then it is not a viable method in its current state. As the bead packed
channels rely on capillary action, based on the interactions of adhesion and cohesion, as the
channel fills with more plasma, it will ultimately reach a point where no more is naturally pulled
through and collected. This point may be reached before the desired volume is collected.
For the bent channel method, there should not be a limit to the amount of plasma that can
be collected, as long as more blood is pumped through. One potential problem that may be
encountered could be clogging of the system, but without a clog, it should continue filtering the
plasma indefinitely. The biggest factor in deciding how effective this method is will likely be the
concentration of red blood cells in the final samples of plasma. Based on the concentrations of
the cells, the plasma sample may not be clinically useful for the tests that are typically performed
on pure plasma. The volume is not expected to be an issue, as the system relies on external
pressure that can be applied to keep the blood continuously flowing through the system.
3.4 Safety Analysis
An inherent risk of working with human blood is exposure to bloodborne pathogens, such
as Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus
(HCV). HIV is a virus that attacks your immune system and can develop into Acquired Immune
Deficiency Syndrome (AIDS). AIDS leaves the body unable to adequately defend against
infections or diseases, and can be fatal. There is no cure for HIV or AIDS, but there are
treatments to control HIV and, if diagnosed early enough, people with HIV can lead a normal,
mostly healthy life.

[18]

Hepatitis B is a viral infection caused by HBV that infects the liver.

10

Hepatitis B can cause acute or chronic infection, the latter putting patients at high risk of death
from cirrhosis of the liver or liver cancer. There is currently no cure for HBV, but there is a
vaccine.

[20]

Similarly, Hepatitis C is a liver disease caused by HCV. Hepatitis C can be acute or

chronic; the former is generally asymptomatic, while the latter can lead to cirrhosis of the liver.
Hepatitis C does not always need treatment because the immune response can often eliminate the
virus. For more serious cases, there are a variety of treatments that can control and cure Hepatitis
C.

[21]

HIV, HBV and HCV are the most common bloodborne viruses, however many other

pathogens exist and could potentially infect anyone exposed to infected blood. There is also a
risk that a sample carries a bloodborne pathogen that has yet to be discovered, which would have
no cure and very minimal, if any, treatment options. The unknown disease could produce any
number of symptoms, including, but not limited to, death.
To minimize risk, all personal protective equipment should be worn at all times,
including gloves, a laboratory coat and safety glasses. All work must be done in a Biosafety
Level II laboratory , with the wet-work taking place inside of a Class II Biosafety Cabinet, both
[22]

to protect the samples from airborne contaminants and to protect the user from exposure. The
[23]

BSC must be cleaned thoroughly with 70 percent ethanol before and after use.
The scientist must be especially careful with the capillaries. The capillaries could easily
puncture gloves or skin. If this happens, the scientist must remove compromised gloves and wash
hands thoroughly. If the capillary penetrates the skin, the scientist must flush the wound with
water for 15 minutes, then immediately contact their primary investigator, and seek medical
attention. The blood will be procured from ZenBio, from which healthy blood will be purchased
for the purpose of this experiment. While the blood is expected to be healthy, there is always a
chance a sample carries a virus unbeknownst to the donor.
Once the experiment is completed, proper disposal of the biological waste is imperative.
Liquid waste must be decontaminated by adding approximately ten percent bleach and leaving it
for at least ten minutes before being emptied into the sink with running water. Any containers
must be emptied into waste containers marked for biohazard. All capillaries must be disposed of
in biohazardous sharp waste immediately after use. Leaving capillaries out after use increases the
risk of incident. Any syringes, pipet tips, or needles used also belong in sharp biohazardous
waste. Hazardous contents will then be autoclaved and disposed of by an outside source.

[24]

For the safety of the scientists, and other laboratory workers, all personnel working with the
11

biohazardous material must be trained properly in safety practices and safe disposal of material
through Northeastern University Environmental Health & Safety. Scientists working with the
blood must be vaccinated for Hepatitis B.
4. Experimental Timeline
Depending on the complexity of the designs, machining the molds can be very timeconsuming and expensive. However, a simple design will be sufficient for the methods described
above. Machining the molds will be outsourced to Stanford Microfluidics Foundry. While
multilayer soft lithography allows for the mass production of microfluidics plates, the process
must be carefully monitored to ensure consistent quality and success. Some molded plates might
not be viable for the experiment and each plate must be carefully examined to ensure that they
have the correct dimensions, even though a certain level of error is acceptable. Creation of the
plates will be outsourced to Stanford Microfluidics Foundry as well. It is expected that all plates
will arrive within three weeks.
Task 1 will be set-up, including sterilizing the work area and ensuring that biohazard
safety measures are in place (20 minutes). After the completion of Task 1, Task 2 will be to
prepare the chips for experimentation by inserting beads into the bead packed chip channels and
running water through each chip to ensure there are no obstructions to flow. Task 3 and Task 4,
the calibration of both the syringe pump and the spectrophotometer can be conducted alongside
Task 2 (60 minutes for each task). These tasks are part of preparation, and sums up to
approximately 80 minutes.
After preparation, the experiment will be executed. Task 5 will be to run all six beadpacked chips simultaneously. At the same time, Task 6 will be to run each bent-channel chip
individually. These two tasks should be completed within 60 minutes. After beginning the
separation of blood on the chips, the first sample of plasma is expected to be collected and ready
for analysis within 10 minutes. Task 7 can be begun, which will be to measure the absorbance of
each plasma sample. All 24 samples can be analyzed within approximately 120 minutes.
Following the completion of the absorbance measurements, the work area must be cleaned and
bio-hazardous waste disposed of (15 minutes). The total experiment execution time is estimated

12

to be 145 minutes. This time falls well below the allotted 210 minutes, leaving room for
unexpected setbacks.

Figure 4: Estimated duration of key tasks

13

5. Cost
Analysis
Table
1: Cost
Analysis
Table

[25-44]

14

Based on Table 1, the total tentative cost for this experiment is $14,152.82, which falls
under the $20,000 budget. The equipment and materials that are listed in Table 2 are considered
and selected based on the experimental protocol factored into preparation, execution, and safety.
Each component is to be obtained by means of purchase, borrowing, or prior possession.
Purchasing may require communication with customer service in order to confirm specifications,
parameters, and expected date of delivery. Borrowing services and equipment from the university
places emphasis on negotiating with faculty and staff in order to select times of use that interfere
as little as possible with other research projects and experiments. Owning certain materials such
as personal protective equipment slightly lightens the cost and dependency on outside sources.
Outsourcing companies for the development of microfluidics plates can be a costly
process. Often times, product development firms charge the highest expense in outsourcing
molds, starting from approximately $50,000 to higher depending on complexity of features. This
estimate more than doubles the available budget. The most practical means of outsourcing is
through either a one to two person shop or at a university facility such as the Cornell
Nanofabrication Facility and the CMB2 facility at LSU, both of which were recommended by
Professor Shashi Murthy. The Stanford Microfluidics is the most cost-effective choice that meets
experimental parameters. The Stanford Microfluidics Foundry is not only able to make the molds
at a very low cost of $175 per mold, but they can also proceed to use these molds to develop the
actual channel chips for the experiment at $30 per chip . This labor saves the team from having
[32]

to perform multi-layer soft lithography or injection molding themselves, a process that

jeopardizes quality of the plates and demands additional costs of materials, such as the injection
polymer .
[32]

The colloidal silica beads, glass beads, PBS, and DI water are used in the development of
the bead-packed mold designs. The composition and size of these beads are based off of the
earlier experiment in which the diameter of these beads enforce the separation of red blood cells
and plasma based on their contrasting sizes. DI water is essential for rinsing any materials, and it
is also used to test for any obstructions in the plates. Using this type of water as opposed to
regular water is very important for experiments, especially those on the microfluidics scale,
because the incorporation of ions such as sodium, calcium, and iron could affect experimental
results

[33-35]

The whole blood unit, isolated red blood cells, and plasma are both purchased from
ZenBio, a company that is FDA licensed and pathogen tested for the following: HBsAG, Anti15

HVC, Anti-HIV-1/2, HIV-1 RNA, HBV DNA, HCV RNA, and Syphilis. The absence of these
pathogens is implemental for upholding safety for the experimenters as well as for those whose
equipment and lab space is being borrowed. .
[37]

The experiment requires a flowrate between 1.0 and 2.0 uL/s which is met by the
programmable syringe pump. This device has a minimum of 0.001 uL/s and a maximum flowrate
at 25.49 uL/s. These parameters are based on the 0.5 uL syringe that is used to pump blood into
the bent channel chips to collect a volume of 0.5 uL .
[39,40]

The microspectrophotometer purchase is primarily based upon meeting the experimental

parameters and secondly dependent on a reasonable cost. The wavelength range of the device is
200-800 nm, encompassing the wavelength of pure plasma used to detect red blood cells, 280
nm. The minimum sample size of the device is 0.5 uL, which is fairly below the collected sample
size of the experiment, 5.0 uL, thus promoting accurate readings. This device and the 0.1mL
cuvettes are both sought out from the same company in order to promote the compatibility
between the cuvettes and the device. In order to avoid the long process of autoclaving, 26
cuvettes are purchased. These cuvettes are for blanking the spectrophotometer with pure plasma,
the simultaneous run of the bead-packed chips, the trials of the bent chips, and the
microspectrophotometer calibration curve .
[41,42]

In order to ensure precise volume collection, six proscopes are purchased. By having
these proscopes work simultaneously for the bead-packed chips, experimenters are able to time
the volume collections by viewing them on expanded computer screen windows, allowing
reliable results that do not simply depend on the naked eye .
[44]

A university laboratory is used for certain equipment and an overall workspace that
accommodates biology-related experiments. Dr. Crams biosafety cabinet and biosafety
hazardous waste disposal will be used for the experiment setting and for removing waste that is
not fit for ordinary laboratory disposal. Experimenters will provide the necessary PPE with the
exception of nitrile gloves, which will be purchased.
6. Broader Impacts
Engineering better medicines has been identified as one of the grand challenges of
engineering. The role of the engineer is to develop faster and more accurate means of diagnosis.
The ultimate goal is to move toward personalized medicine. Personalized medicine refers to the
16

system of prescribing therapeutics tailored to a patient's specific genome . A particular patient

[1]

may overexpress a particular gene, which could confer resistance to an antibody that typically
could treat this patients condition[1]. Likewise, the same overexpression could lead to the patient
being more receptive to a different treatment option. If expression of biomarkers could be tested
before diagnosis and treatment, there might be another treatment option, or the traditional
treatment could be prescribed in conjunction with a therapeutic that represses the overexpressed
gene. At this time, these kinds of tests are too costly and time-consuming to be feasible, so often
patients end up on drugs which do not help them. The goal is to create efficient and affordable
processes to make personalized medicine a reality.
As the health care system progresses toward personalized medicine, plasma being quickly and
effectively separated from blood cells will allow testing for biomarkers to be conducted more
rapidly. Protein levels in plasma are already being tested as a means of detecting many diseases.
Increasing the efficiency of separating the plasma from blood cells would also increase the speed
at which a laboratory could process a large number of samples, resulting in a quicker diagnosis
and potentially improved patient prognosis. Currently, rapid separation is often conducted via
centrifugation, which can cause contamination from blood cell lysate. This experiment could
demonstrate more effective means of separation without compromising speed or cost.
As the process is more refined and optimized, the microfluidic blood separation
techniques could potentially greatly increase the speed and efficiency of plasma separation and
contribute to these improved outcomes. After further experimentation, the process should
become quicker, more accurate, and less expensive, allowing for more efficient plasma collection
resulting in more personalized therapies.

17

7. References
[1] "Engineer Better Medicines." Grand Challenges -. N.p., n.d. Web. 13 Oct. 2015.
[2] "Medicines in Development." Medicines in Development. PhRMA, n.d. Web. 13 Oct. 2015.
[3] "PhRMA & LLS: More than 240 Medicines in Development for Blood Cancers." PhRMA &
LLS: More than 240 Medicines in Development for Blood Cancers. PhRMA, n.d. Web. 13 Oct.
2015.
[4] "DNA Sequencing Lays Foundation for Personalized Cancer Treatment." - The Elizabeth H.
and James S. McDonnell III Genome Institute at Washington University. Washington University
in St. Louis School of Medicine, n.d. Web. 19 Nov. 2015.
[5] "What's New in Acute Myeloid Leukemia Research and Treatment?" What's New in Acute
Myeloid Leukemia Research and Treatment? The American Cancer Society, 9 Dec. 2014. Web.
13 Oct. 2015.
[6] Basu, Debdatta, and Rajendra Kulkarni. "Overview of Blood Components and Their
Preparation." Indian Journal of Anaesthesia. Medknow Publications & Media Pvt Ltd, n.d. Web.
19 Nov. 2015.
[7] "Microfluidics for Cancer Cell Detection and Diagnosis" National Center for Biotechnology
Information. U.S. National Library of Medicine, n.d. Web. 14 Oct. 2015.
[8] "HMS." MICROFLUIDICS/MICROFABRICATION. Harvard Medical School, n.d. Web. 13
Oct. 2015.
[9] "Plasma." American Red Cross, n.d. Web.
[10] ["Plasma Protein Tests." Healthline, n.d. Web. 22 Nov. 2015.]
[11] "Blood Tests to Detect Inflammation, Protein in Blood | Patient." Patient. N.p., n.d. Web. 22
Nov. 2015.
[12] [Kroh, Evan M., Racheal K. Parkin, Patrick S. Mitchell, and Muneesh Tewari. "Analysis of
Circulating MicroRNA Biomarkers in Plasma and Serum Using Quantitative Reverse
Transcription-PCR (qRT-PCR)." (2010): n. pag. Europe PubMed Central. Web.]
[13] Blattert, C., et al. "Microfluidic blood/plasma separation unit based on microchannel bend
structures." Microtechnology in Medicine and Biology, 2005. 3rd IEEE/EMBS Special Topic
Conference on. IEEE, 2005.
[14] Perlman, Howard. "Capillary Action." , from USGS Water-Science School. U.S. Geological
Survey, n.d. Web. 19 Nov. 2015.
[16] Shim, Joon S., and Chong H. Ahn. "RAPID ON-CHIP BLOOD/PLASMA SEPARATOR
USING HETERO-PACKED BEADS AT THE INLET OF MICROCHANNEL." (n.d.): n. pag.
Print.
[17] "Beer's Law - Theoretical Principles." Beer's Law - Theoretical Principles. Shaffield Hallam
Unversity, n.d. Web. 11 Dec. 2015.
[18] Motrescu, Iuliana, Servilia Oancea, Alina Rapa, and Anton Airiness. "Spectrophotometric
Analysis of the Blood Plasma for Different Mammals." ResearchGate. University of Agricultural
Sciences and Veterinary Medicine, Jan. 2006. Web. 24 Nov. 2015.
[19]"What Are HIV and AIDS?" What Are HIV and AIDS? | AVERT. ADVERT, 01 May 2015.
Web. 08 Dec. 2015.
[20] "Hepatitis B." WHO. World Health Organization, July 2015. Web. 08 Dec. 2015.
[21] "Hepatitis C." WHO. World Health Organization, July 2015. Web. 08 Dec. 2015.
[22]Bio-Safety Training 2 Biosafety, Office of Research Assurances, Washington State
University. Web. 30 Nov. 2015.

18

[23] "Safety and Health Topics | Bloodborne Pathogens and Needlestick Prevention." Safety and
Health Topics | Bloodborne Pathogens and Needlestick Prevention. Osha, n.d. Web. 19 Nov.
2015.
[24]Facts about Biohazardous Medical Waste Treatment. Phoenix, AZ: Arizona Dept. of
Environmental Quality, 2001. Stanford University. Web.
[25] "Human Blood Products." Human Blood Products. ZenBio, Inc., n.d. Web. 17 Nov. 2015.
[26] Spectrophotometer. Amazon. N.p., n.d. Web.
[27] Bourzac, Katherine. "Ten-Minute Blood Test | MIT Technology Review." MIT Technology
Review. N.p., 17 Nov. 2008. Web. 17 Nov. 2015.
http://www.technologyreview.com/news/411193/ten-minute-blood-test/page/2/
[29] "Human Blood Products." Human Blood Products. ZenBio, Inc., n.d. Web. 17 Nov. 2015.
[30] Spectrophotometer. Amazon. N.p., n.d. Web.
[31] Bourzac, Katherine. "Ten-Minute Blood Test | MIT Technology Review." MIT Technology
Review. N.p., 17 Nov. 2008. Web. 17 Nov. 2015.
http://www.technologyreview.com/news/411193/ten-minute-blood-test/page/2/
[32] "Pricing Information." Stanford University. Web. 11 Dec. 2015.
[33] Monodisperse Silica Microspheres. Cospheric LLC, 2015. Web. 24 Nov. 2015.
[34] "Glass Beads Abrasive Blasting Media." Kramer Industries Glass Beads. Kramer Industries,
2015. Web. 24 Nov. 2015.
[35]"Why Use Deionized Water?" LIVESTRONG.COM. LIVESTRONG.COM, 20 July 2015.
Web. 11 Dec. 2015.
[36] "Phosphate Buffered Saline, 10X Solution, Fisher BioReagents:Chemicals:Buffers."
Phosphate Buffered Saline, 10X Solution, Fisher BioReagents:Chemicals:Buffers. Fisher
Scientific, 2015. Web. 25 Nov. 2015.
[37] "Human Blood Products." Human Blood Products. ZenBio, 2015. Web. 25 Nov. 2015.
[38] "Disposable Syringe." Amazon, 2015. Web. 25 Nov. 2015.
[39] "NE-1000 Programmable Single Syringe Pump." Syringe Pump. New Era, 2015. Web. 25
Nov. 2015.
[40] "10 L, Model 701 N Micro SYR Pipette, 0.2 - 3 L." 10 MicroL M Model 701 N Micro
SYR Pipette 02 3 MicroL. Hamilton Company, 2015. Web. 25 Nov. 2015.
[41] "Micro Pipette Disposable Tip, PTFE 100 Pack." Micro Pipette Disposable Tip PTFE 100
Pack. Hamilton Company, 2015. Web. 25 Nov. 2015.
[42] "Professional Micro-Spectrophotometer Nano-100 Range: 200~800nm." Joyfay.com.
JoYFay, 2015. Web. 25 Nov. 2015.
[43] "Kimberly-Clark Model KC500 Nitrile Powder Free Exam Gloves, Disposable, Purple."
Amazon.com: : Health & Personal Care. Amazon, 2015. Web. 25 Nov. 2015.
[44] "Top Categories." B&H Photo Video Digital Cameras, Photography, Camcorders. Web. 11
Dec. 2015.
[45] Hartman, Jeremy. Design Project Proposal 15 Oct. 2015.

19

Appendix A: Experimental Procedure

1. Design four separate molds in AutoCAD based on the four different types of plates
needed: one mold for the bent-channel design with a 100um main channel and a 20um
side channel extending at 90 degrees from the bend, another of the bent-channel design
with a 200um main channel and 40um side channel extending at 90 degrees from the
bend, a mold for the bead packed channel design with a 100um opening to the capillary,
and a mold for the bead packed channel design with a 75um opening to the capillary.
2. Order molds for chips and company will use multilayer soft lithography to create chips
according to a procedure described in the literature from Stanford Microfluidics Foundry.
3. Receive chips from Stanford Microfluidics Foundry
4. Calibrate the single-syringe pump for appropriate flowrates, using the measurements of
the time required to pump out the known volume.
5. In the plates made from the bead-packed mold designs, seal the opening of the channels
with microbeads using the method described in a similar experiment.
6. Rinse the channel with DI water, then insert 100 micron diameter beads and pressure pull
through to block the channel opening using syringe.
7. Draw PBS through to seal these beads in place
8. Draw through 10 micron diameter beads to block the channel on top of the larger beads
9. Draw PBS through to seal these smaller beads in place
10. Test all plates received from the Stanford Microfluidics Foundry to make sure they
function as expected, by attaching chip to bottom plate and running de-ionized water
through the channels at a known flow rate using the calibrated single-syringe pump to test
for obstructions in each chip
11. Dry out chips.
12. Sterilize chips with 70% ethanol.
13. Calibrate the spectrophotometer for 100 percent transmittance by blanking the equipment
with a 5uL sample of pure plasma at a wavelength of 280nm , the wavelength used to
detect red blood cells.
14. Prepare 5uL standards of 15 percent, 30 percent, 45 percent, 60 percent, 75 percent and
100 percent isolated red blood cells in pure plasma
15. Measure 5uL standards of 15 percent, 30 percent, 45 percent, 60 percent, 75 percent and
100 percent isolated red blood cells in pure plasma on the spectrophotometer.
16. Attach chips to bottom plate and load the syringe pump with a syringe containing the
desired amount of blood, then attached through the opening into system.
17. On the bead packed channel chips inject 40uL of whole human blood into the reservoir at
the opening of the channel using a syringe and start the timer. Use blood containing 5
percent heparin to prevent clotting of blood inside the channels.
18. Monitor flow on the bead packed chips until plasma has accumulated in collection
reservoir up to the mark for a volume of 5uL, then stop the timer. Record time for each
chip in Table A-1.
19. On the bent channel chips inject 80uL of whole human blood into the opening of the
channel at the selected blood flowrate using the syringe pump, and start the timer. Run
one chip of each channel size at each of the following pump flowrates; 0.5uL/s, 1uL/s,
[16]

[18]

A.1

and 2uL/s. Use blood containing 5 percent heparin to prevent clotting of blood inside the
channels.
20. Monitor flow until plasma has accumulated in collection reservoir up to the mark for a
volume of 5uL, then shut off syringe pump and stop the timer. Record time and pump
flowrate setting for each chip in Table A-1.
21. Calculate the flowrate of plasma collected for all chips based on time required to fill the
reservoir to 5uL and record result for each chip in Table A-1.
22. On each bent channel chip, repeat this method three times for each configuration,
repeating the same pressures and volume added on all of bent channels chips. Wash the
chip with 70 percent ethanol between each run.
23. Run the bead packed chips concurrently for efficiency because of the length of time
capillary action takes to separate the plasma. Run each design under the same condition,
adding the blood and applying no pressure. This should result in each measurement being
taken 3 times.
24. After each run on a chip, of either design, transfer the 5uL of plasma collected in the
reservoir into a cuvette, using a syringe.
25. Test the purity of the samples in the cuvettes using the spectrophotometer. Blank
spectrophotometer with pure plasma in an identical cuvette prior to running each sample,
and record readings in Table A-1 then compare to the calibration curve to quantify the
purity of the sample collected and whether any red blood cells are still present.
26. Calculate concentrations from the percent transmittance using Beers Law, and use
concentrations of red blood cells for comparison of the purity of samples produced by
different methods. Record concentrations in Table A-1
Appendix B: Raw Data Tables
Table A-1: Data Table Template for Raw Data Collected
Chip
number &
Design

Blood
Flowrate
(for bent
channel)

Erro
r

Trial
number

Tim
e

Erro
r

Volume
collected

1
Bent
Channel
20um

0.5uL/s

5uL

0.5uL/s

5uL

0.5uL/s

5uL

2
Bent
Channel
20um

1 uL/s

5uL

1 uL/s

5uL

1 uL/s

5uL

---------

------ 1

5uL

Erro
r

Flowrate
of Plasma

Erro
r

A.2

Bead
Packed
100um

---------

------ 2

5uL

---------

------ 3

5uL

etc.

Chip
number

Trial
number

Avg Error %
Flow
Transmission
rate

Error Avg %
Transmission

Error

Concentration
of RBCs

2
3
2

1
2
3

etc.
Appendix C: Sample Calculations
Flowrate of blood = 1uL/s
Volume of serum collected = 5uL
Time for collection = 25sec
Error = of smallest unit of measure
Flowrate of Plasma= Volume collected/time for collection=
Error=
Avg Flowrate of Serum= avg of flowrates for chip 1 =

5 uL
25 s

= 0.20 uL/s

0.20+0.21+0.19
3

= 0.20 uL/s

Error =
% Transmittance concentration of red blood cells in sample
Beers Law to find Molar Absorptivity:
A=bc

A.2

A
= bc
A=75%=0.75
b=1mm
c=1.333g cells/ mL blood
=

A
=
bc

0.75
g cells
( 1 mm ) 0.5625
mL blood

= 1.33

mL blood
mmg cells

A.2