Automated, Accessible, and

Affordable: Development of a
Novel Flu Diagnostic
Design Proposal
Jessica A. Wang
PI: Paul Yager, Ph.D.
Department of Bioengineering
University of Washington
June 6, 2014

This capstone project proposes to create a novel paper diagnostic for the detection of influenza viruses from a
nasal swab sample. This will be achieved using a dynamic paper network with switching and valving
mechanisms designed to perform complex immunoassays. Automation techniques will be incorporated such
that the final device will be accessible to users with no laboratory training. Although this capstone considers
only the integration of Influenza A nucleoprotein assays, the device produced from this work will have the
capacity to be integrated with assays for other pathogens. This capstone will provide a design that can be
adapted to create rapid and affordable diagnostics in response to new epidemics and for the promotion of
public health.

Table of Contents
Background and Significance ............................................................................................................................ 2
Statement of Problem and Solution ................................................................................................................ 2
Previous Approaches ..................................................................................................................................... 3
Preliminary Data ............................................................................................................................................ 4
Consequences of Success ............................................................................................................................. 6
Ethical and Social Issues ............................................................................................................................... 6
Economic Issues ............................................................................................................................................ 7
Legal and Regulatory Issues .......................................................................................................................... 7
Plan of Work .................................................................................................................................................... 10
Specific Phases ........................................................................................................................................... 10
Design Strategy ........................................................................................................................................... 11
Phase 1: Development of Paper Network and Tracking Flow with Colored Reagent Simulants ................ 11
Phase 2: Integration of Nucleoprotein Assay Reagents Including Label and Capture Antibodies, Wash
Buffers, and Gold Nanoparticles for Detection .......................................................................................... 12
Phase 3: Integration of Swab Delivery and Lysis ...................................................................................... 14
Engineering Design Standards ..................................................................................................................... 15
Key Personnel.............................................................................................................................................. 15
Equipment, Facilities, and Resources .......................................................................................................... 15
References ...................................................................................................................................................... 17
Appendix ......................................................................................................................................................... 18
I.

Request for Proposal (RFP) .................................................................................................................. 18

II.

Concept Sheet ...................................................................................................................................... 19

III. Milestones and Timeline Table .............................................................................................................. 20

Background and Significance

Statement of Problem and Solution
A type of flu, Influenza A, killed 50 million people worldwide during the 1918-1919 pandemic, most of which
were previously healthy young adults [1]. The uncontrolled spread of deadly flu viruses can be devastating,
and yet we still have not developed adequate technology to prevent future outbreaks from spreading. Rapid
diagnostics on the market provide means for diagnosing users with either type A or B influenza, but these tests
are complicated and require special certification or laboratory training. Usually these tests also require a
Clinical Laboratory Improvement Amendments (CLIA) certificate of waiver [2]. Ease of use is a problem when
we consider the needs of the at-home user or official personnel in response to an emergency outbreak.
Knowing if a person is infected with influenza and, further, what strain of influenza is causing the infection,
provides useful information for diagnosis and containment of the disease. Type A influenza is traditionally the
strain known to cause wide-spread pandemics that affect both people and animals [8]; hence, A/B influenza
typing is of particular interest to health care workers worldwide.

A device that integrates sample preparation and Influenza A detection into a one-step-activated model would
be more accessible to the everyday user for preventing the spread of virus and keeping contagious individuals
in their homes. In addition, this technology has potential to be used by immigration control agencies to prevent
spread of disease worldwide. The Yager Lab is in the process of developing an integrated paper diagnostic
that provides a means to combat future influenza pandemics. My focus will be to develop automation
technologies for the final diagnostic that reduce the number of user steps. The processes I will simplify include
sample preparation, which involves lysis of the virus from a nasal swab, activation of the influenza
nucleoprotein (NP) assay, and running the assay in a diagnostic paper strip. My final device will meet design
specifications outlined in Table 1.

Design Specification
Number of user steps
required for activation of
device
Run time of device
Limit of Detection (LOD)

Specific detection

Target values and tolerances

1, max 2

Validation Tool
Counting of steps while
performing activation

< 30 minutes
10 pM of viral protein (NP)

Timer
Trials with varying
concentrations of viral protein
(NP) and examination for the
presence of test lines
Imaging of the resulting test
strips using a scanner and
color intensity analysis of the
resulting detection lines using
color intensity quantification
program written in MATLAB.

For a test that should run positive

(run with a sample containing NP):
both detection line and control line
should turn a red color at least 3x
the intensity of background.
For a test that should run
negative: detection line should not
be visible, but control line should
turn a red color at least 3x the
intensity of background.
Table 1. Acceptance criteria for Development of Automated, Easy to Use Flu Nucleoprotein Detection

Previous Approaches
In the past couple of decades, point-of-care paper diagnostics have become increasingly popular for use at
home and other applications outside of the clinic. A well-known lateral flow test is the pregnancy test strip, on
which a female user urinates and can receive information within minutes about whether or not she is pregnant.
The test detects the presence of the human chorionic gonadotrophin (hCG) hormone using a capture antibody,
to plant the hormone on a detection line, and a labelling antibody, to make the presence of the hCG obvious [3].
This paper test format has now been implemented into a variety of diagnostics for diseases including the
influenza virus.

Accessibility remains one of the most difficult challenges in influenza testing. Traditionally, influenza testing is
done in a medical or public health laboratory and can take days to process. A physician or nurse would take a
nasal swab or nasal wash from a patient and send the sample to the laboratory, where highly specialized
technicians will perform one of a variety of tests. These tests include viral culturing, polymerase chain reaction
(PCR) testing, immunofluorescence assays, and serologic testing [9]. In the past years, commercial, rapid
lateral flow tests have become increasingly popular as they enable users to conduct flu testing in clinics
outside of medical laboratories. For instance, one commercially available flu diagnostic is the Alere Influenza
A&B test, which is a rapid diagnostic that uses a detection strip to detect influenza from a nasal sample [4].
Using Aleres method of detection involves multiple steps that must be carried out with both hands, including
stirring the swab in an open container of liquid and manually inserting the paper detection strip into a vial with
the sample. A survey of the Centers for Disease Control and Preventions Rapid Influenza Diagnostic Tests
Table updated in 2013 shows that many of the commercial devices available have similar methods for use with
independent components for mixing the sample and initiating the test [2]. Although lateral flow strips have
enabled testing to be done outside of laboratories for more immediate point-of-care diagnosis, these devices
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still lack methods that integrate sample preparation and test initiation simply into a diagnostic. It is also of note
that many physicians are opting not to use rapid lateral flow tests in the clinic due to their lack of sensitivity [12],
an issue that can be address by being able to do more complicated assays in paper.

The Yager Lab has developed a preliminary solution to this issue in the format of a two-dimensional folding
card paper diagnostic. In particular, Fu et al. demonstrated the use of this multi-leg paper platform for the
detection of malaria [5]. The device uses a series of glass fiber pads that have all had reagents dried down
onto them. Immediately before folding the card, these pads are rehydrated with water, which allows reagents to
flow into the paper strip. The shape and spatial organization of the pads with respect to the paper makes it
such that the reagents are delivered sequentially in the desired order. The capacity to add multiple reagents
during the testing process allowed for the addition of amplification reagents, which significantly improved
sensitivity of these diagnostics. A model of this format is demonstrated with food colored water in Figure 1. The
Yager Lab has also successfully applied a similar version of this folding card to a flu hemagglutinin protein
assay, but due to the fact this research has not yet been published, details about those experiments are being
kept confidential and not included here.

Although the folding card test is automated, the major downside to this method is that preparation of the card
still requires pipetting fluid onto each pad before folding. In addition, the sample still has to be in prepared in
lysis buffer external to the device before pipetting. My project will work to simplify this process and create an
easy-to-use interface for an automated flu diagnostic that also allows for the addition of several reagents for
complex assays.

Flow

Figure 1. Folding card format that was implemented in the malaria diagnostic [5]. The card delivers first the red
and then the green reagents to a detection line right before the pad.

Preliminary Data
The assay that will be implemented in the diagnostic is a flu nucleoprotein (NP) assay. NP resides on the
inside of the influenza virus and is distinct depending on if the influenza is of type A or B. The Yager Lab and
our collaborators from PATH have been working on an assay that detects for NP from Influenza A. The
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underlying concepts behind this assay are outlined in Figure 2. We currently have knowledge of a NP assay
that works consistently on nitrocellulose paper in a dipstick format, where a paper strip spotted with a detection
line is manually dipped in reagents pipetted into a 96-well plate. We have established the limit of detection
(LOD) for our assay to be within the clinical range of virus concentration a person must have to be considered
diagnosed. Although others in the lab continue to research methods for decreasing the LOD for greater
sensitivity, I will be using an assay with a LOD of 10 pM and adapt my work as necessary to implement new
assays with better LOD as they develop.

With regards to sample preparation, the lysis protocol to retrieve flu nucleoprotein has been well established.
The lysis solution currently being used in the lab to release NP is a combination of phosphate buffered saline
with Tween (PBST) mixed with 5% of a non-ionic detergent, TritonX-100. In addition, the solution has 4%
bovine serum albumin protein (BSA), which helps preserve the stability and structure of the proteins. This
solution is directly mixed with a viral sample to expose NP for the assay. One difficulty we have identified in the
lysis solution is that BSA has to be kept cold and used within 4-5 days. Further work has to be done in the
development of preserving and storing BSA in paper devices.

The Yager Lab has also been experimenting with different methods for transferring sample from a swab into a
device in another project in the lab called MAD-NAAT [11], which is for the development of a diagnostic for
pathogenic nucleic acids. In the current stage of development, the MAD-NAAT team has characterized many
approaches for integrating lysis and sample preparation into a simple process. The design for the user
interface they are developing allows for easy insertion and rotation of a swab for release of the sample in a
lysis buffer. This swab case is also in direct contact to the diagnostic paper strip. Although the lysis conditions
for nucleic acid amplification and the flu nucleoprotein assay are quite different in terms of buffer composition
and temperature requirements, the technologies that the MAD-NAAT team has iterated through will serve as a
starting point in my development of a swab case for the influenza diagnostic.

Previous work I have done related to this project (currently pending publication) include the creation and
characterization of a variety of dynamic paper networks that allow for controlled reagent delivery. The majority
of the networks I have developed involve time-activated sponges that will expand upon contact with water and
force either contact or separation between two paper strips, thus controlling where fluid flows within the paper
strips. In this most recent application of these techniques, I have developed a device that mimics the delivery
of fluids in the folding card design but does so without the need for pipetting fluids before activation. In my
design, two glass fiber pads with dried reagents are placed above sponges which are touching a glass fiber
wick running along the bottom of the device (Figure 3). These glass fiber pads are situated directly underneath
a paper strip, which, in the final device, will be spotted to have detection and control lines. At the activation of
the device, the ends of a paper strip and glass fiber timing wick are dipped into a well such that fluid starts to
5

flow through them. The length of the glass fiber wick is designed such that there is a sufficient delay between
the start of the test and the moment when the liquid reaches the first sponge. This is to allow the fluid in the
paper to reach the detection area before addition of any reagents. When the sponge expands and pushes the
first glass fiber pad to contact with the paper, the fluid in the paper rehydrates the contents in the pad and
carries the reagents downstream. The length of glass fiber between the first and second sponge is also
designed to create a long enough delay to allow reagent from the first pad to reach the end of the detection
area before introduction of the second reagent (Figure 4). Although my work thus far has been limited to food
dye simulating reagents, my device demonstrates automatic delivery and requires no user intervention after
the initial activation.

Consequences of Success
The outcome of my project will be the creation of a point-of-care diagnostic that can perform the flu NP assay
in an encapsulated and automated format. The device will activate with ideally one or maximum two user steps
from the introduction of a swab with sample to the device. I will run multiple trials of my device to insure that
the assay performs and maintains the sensitivity and specificity it had originally in the lab bench top form.

My technology is important because it will address a current gap in the field of rapid influenza diagnostics for a
test that is accessible and can be used easily by a person with no special training. It will be the first of its kind
to address the need for automating and simplifying these tests. Easy-to-use paper diagnostics have the
potential to be implemented in clinics, airports, and even peoples homes as they develop to become userfriendly and safe for anyone to use. While my diagnostic will be developed specifically for influenza, the
underlying technology in my device will have the capacity to be adapted to assays for other diseases. In this
way, my technology proposes to increase our ability to cope with future pandemics and maintain public health
by providing rapid means of diagnosis.

Ethical and Social Issues

In the process of engineering new medical technologies, engineers have to be mindful of ethical and social
issues that may limit the impact of their designs. In the case of the project proposed here, it is important to
consider accessibility as both a technical and an ethical issue. As with any diagnostic, there is the potential that
the information a medical device provides to either a patient or health care provider carries greater
ramifications than simply information about ones health. For instance, if in the future this influenza diagnostic
were to reveal that a patient carries an extremely contagious and deadly strain of flu, whether or not this
information should be disclosed to anyone other than the patient is an ethical issue that also can affect the
welfare of others in the vicinity. There may be other factors to consider as well, such as whether or not it would
be acceptable to detain a contagious person or require the contagious to wear indicative garments such as
face masks. Another ethical issue is the question of who should and would be required to have a test.
Admittedly, the process of acquiring a nasal swab sample is quite invasive, and so there may be individuals or
cultures that consider being required to take this test a violation of their autonomy. These are the issues that I
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will have to take into consideration as an engineer in the development of my device in order to best suit the
product for realistic intended use.

Economic Issues
As with any device looking to be implemented in the market, there are important economic issues to consider
in the development of the proposed diagnostic. Although the devices will be inexpensive to manufacture and,
thus, affordable for purchasing, there are additional costs associated with the implementation of any medical
diagnostic, especially if we try to implement this technology in places that do not already use diagnostics on a
regular basis. There would likely be a cost involved in educating and advertising to the general populace on the
importance of early diagnoses and the usefulness of at-home testing to keep friends and family healthy. To
supplement this, there would also have to be education on the appropriate uses for the device such that
worried parents will not be over-testing their children or attempting to use them for a purpose for which they are
not intended. Another issue we have to consider is what the price to buy a test should be if it is to be truly
accessible to all users. It may make sense to sell the same test at different prices based on how developed the
economy is at that location. One such company doing this is Cepheid, which tends to sell their medical
devices for more in developed countries and less in lower resource areas [13]. Overall, in order for there to be
a positive return on investment in the implementation of these diagnostics, the participation by a large number
of people is required, but when this diagnostic is successfully implemented in peoples homes and in clinics,
this technology can help improve and save the lives of many.

Legal and Regulatory Issues

The most important regulation is the fact that because this diagnostic is intended for human use, it will have to
be inspected and approved by the Food and Drug Administration (FDA) before it can be sold on the market. In
addition, there are potential intellectual property issues that may inhibit the licensing of the intended product.
For instance, the paper and plastic materials used to manufacture the device are made by outside companies
that may or may not allow for the use of their materials in the creation of this diagnostic without a substantial
fee. There is also the issue of ownership with regards to the assay physically implemented in the device. The
assay that I will be implementing was developed in part in collaboration with PATH, and so they would be
entitled to a portion of the royalties for the device. The same would be true for any other assay implemented in
this diagnostic. Finally, this research in its entirety will be conducted at the University of Washington, and so
the university will also hold rights to the developments I make in this project.

Figure 2. Cartoon depicting basics of NP assay. The assay will happen in 3 main steps. Step 1 shows the
capture antibody pre-spotted onto the paper, creating a detection line. Step 2 shows NP being passed over
and captured by the antibody. Step 3 shows a detection antibody conjugated with a gold nanoparticle passing
over the detection line and binding to NP. The aggregation of gold nanoparticles makes the detection line
visible and indicates the presence of specific flu NP in this sample. My assay will be for the detection of NP
from Influenza A.

Detection line of capture

antibodies will go here

Control line that captures

gold nanoparticles will go
here (to verify detection
reagents flowed through)

Figure 3. Glass fiber pusher device (before activation). The design has two glass fiber pads with dried reagent
(colored food dye in this case) underneath a paper strip. The pads are held down by a small flap of plastic with
adhesive. The glass fiber timing wick feeds into sponges underneath both flaps which expand to lift the pads to
the paper. The reagents then flow downstream to a detection line and a control line. A positive test will result in
a both a colored detection and control line. A negative test will result in just a colored control line. Failure to
create any colored lines indicates a defective test.

Figure 4. Time lapse demonstration of sequential and timed reagent deliveries to a detection line. Fluid is
flowing through the paper strip and so the pads rehydrate the food dye when they meet the paper. This causes
the food dye simulating reagents to flow downstream.

Plan of Work
Specific Phases
The purpose of this project is to create a rapid diagnostic that automates the flu nucleoprotein (NP) assay and
reduces the user process to one or two simple steps. To maximize accessibility and affordability, this rapid test
will be done in paper. The assay we are automating includes a virus-lysing step, the introduction of the sample
to a detection strip, multiple additions of antibody reagents, and the production of colored detection and control
lines by addition of gold nanoparticles. I will design a paper-based diagnostic for the detection of NP that: a)
integrates the NP assay into an automated module, b) performs rapidly, and c) is easily accessible to
personnel with little laboratory training. I plan to develop the project following three main phases: 1)
development of the paper network using valving and switching techniques, 2) integration of the NP assay into
the paper network, and 3) integration of the preliminary sample preparation steps, including lysis of viral
sample and sample delivery.
Phase 1: Development of Paper Network and Tracking Flow with Colored Reagent Simulants
The purpose of Phase 1 is to develop the underlying paper network that will be used in the final diagnostic. The
paper network must deliver a series of fluids and reagents to a designated spot for a detection line on a
nitrocellulose paper strip. The delivery of reagents has to be in the correct order, which will be determined by
tracking the various colored fluids within the device. The whole process must finish in 30 minutes for the test to
be considered practical and rapid. A series of sponge activated paper valves with timing wicks and glass fiber
pads carrying reagents will be arranged specifically to achieve this goal. When Phase 1 is complete, we will
have created a paper network that automatically delivers a series of colored simulant reagents to a detection
strip.
Phase 2: Integration of Nucleoprotein Assay Reagents Including Label and Capture Antibodies, Wash
Buffers, and Gold Nanoparticles for Detection
The purpose of Phase 2 is to incorporate the reagents and buffers for the NP assay into the paper network
designed in Phase 1. The NP assay that I will integrate has been established on the bench top, and my design
will adapt in order to optimize the performance of the assay within my device. Success in this step will be
determined by the appearance of a colored control and detection lines formed by the capture of labelling
antibodies, NP, and gold nanoparticles. Tests will also be run with buffer without NP as a negative control and
should produce only a colored control line. Specific reagent amounts and wash steps will be tuned until we
achieve detection lines that display color intensity of at least 3x the background color of the paper. When
Phase 2 is complete, we will have a device that performs an automated form of the NP assay from samples of
recombinant NP.
Phase 3: Integration of Swab Delivery and Lysis
The purpose of Phase 3 is to incorporate lysis and swab delivery of a whole virus sample to the device. We will
do this by first testing that NP obtained from lysing whole virus will still result in the formation of clear detection
lines with a color intensity 3x that of the background. After verifying that delivery by pipette yields detection
10

lines, we will create influenza virus samples using a nasal swab, which will then be used to deliver the sample.
Because swab delivery of a sample is different from simply providing the sample in direct liquid form, this
phase will involve designing and testing a swab case in which the swab will be inserted and soaked in order to
remove the sample. This swab transfer case will first be tested with water to ensure that it can store liquid and
release it onto a paper strip upon activation. It will then be tested for capacity to store lysis solution, allow for
insertion of swab with virus sample, and deliver the lysed virus to paper. The device will also be tested with
lysis solution without viral sample as a negative control. When Phase 3 is complete, we will have a user
interface that is compatible with the lysis buffer, serves as the site for lysis of a virus sample deposited from a
swab, and can be attached to the paper network devised in Phase 2, creating a simple sample delivery and
test activation module for the NP assay.

Design Strategy
In this section I will lay out the approach, the deliverables, and the anticipated outcomes for each of the three
phases I have identified. A visual schematic of my engineering design process is outlined in Figure 5. A table
outlining the timeline for this project is in Appendix III at the end of this document.
Phase 1: Development of Paper Network and Tracking Flow with Colored Reagent Simulants
Approach
In the development of the automated paper network, my goal will be to develop a device that delivers multiple
fluids and reagents to a test strip sequentially using paper switches and valves (developed previously in prior
work, yet to be published). Test devices will be built by stacking layers of poly(methyl methacrylate) (PMMA,
McMaster-Carr, Elmhurst, IL) of various thicknesses and 10 mil Mylar sheets with adhesive backings (Fralock,
Valencia, CA). Embedded within these layers of plastic will be glass fiber GR8975 (Ahlstrom, Helsinki, Finland)
timing wicks, compressed cellulose sponges (Sponge Producers Company, St. Louis, MO), and plastic-backed
nitrocellulose (NC) paper HF135 (Millipore, Billerica, MA). These components will be arranged in a fashion that
allows for timed fluid flow down a main paper strip, on which the detection reagents will be planted. A cellulose
pad CFSP223000 (Millipore, Billerica, MA) for wicking will be placed in contact with the NC paper to facilitate
capillary action through the paper. To simulate the rehydration of various reagents onto a paper strip from
glass fiber pads, food-colored trehalose sugars will be dried onto the pads as antibody and gold nanoparticle
simulants. Trehalose sugars have been shown to help preserve protein structure when integrated with the
dried reagents [10] and so will be included in these simulations in the drying process to provide a more
accurate model. Designs will be drawn using the computer-aided design software Draftsight (Dassault
Systmes, Vlizy-Villacoublay, France), and pieces will be cut using a CO2 laser (Universal Laser Systems,
Scottsdale, AZ). Data will be collected in the form of time lapse pictures taken 10 seconds apart using a
Logitech Pro 9000 webcam (Logitech, Newark, CA) and the program HandyAvi (AZcendant, Tempe AZ). The
resulting images will be compiled and compressed into videos using ImageJ (National Institutes of Health,
Bethesda, MD). Analysis of the movement of colored reagents through the device will be done using an
already developed color intensity quantification program written in MATLAB (MathWorks, Natick, MA).
11

Because the goal of this phase is to develop a network that transports reagents through the device in a fashion
that mimics an assay, the various food colors will be used to trace the reagents in the videos created. The
dimensions and placement of components within each test device will be adjusted depending on the previous
model in order to reach our target fluid flow pattern; that is, a device that delivers the colored reagent simulants
in the correct order and in the timing that closely mimics what would be done for the assay manually on the
bench top dipstick. Our color intensity quantification MATLAB program will be used to trace fluid flow within the
paper. Multiple trials will be conducted to affirm and assess the consistency of results.
Deliverables
The main deliverable of Phase 1 is a dynamic paper network capable of delivering both liquid and dried
reagents in the correct order and timing that would be required to run the NP assay. The method of analysis
will enable the collection of flow rate and fluid volume data, which would then allow us to characterize and alter
aspects of the design that can change these factors.
Anticipated Outcomes
To control the order in which reagents are delivered to the detection line, one current design incorporates a
series of sponges that are used to press glass fiber pads up to the surface of a paper strip. The fluid in the
paper rehydrates the glass fiber pads and releases dried reagents. The order in which we design the sponge
valves to activate will have to be given careful consideration because the order of sponge activation is not
necessarily synonymous with reagent delivery. I anticipate that we will need to perform many iterations of this
device in order to come up with a design that delivers the reagent simulants in a manner most similar to the
labs dipstick form of the NP assay. One difficulty we may encounter in assessing success of this is that the
MATLAB color intensity quantifier program has never been used to analyze a device that has more than one
colored fluid flowing through the region of interest at once. I anticipate there will be challenges in setting
standards for conducting this analysis, but not problems that cannot be fixed by working closely with the
graduate student who created the program. If this method does not work, an alternative method I am
considering is manually splitting the video feed of the device into red, green, and blue channels using ImageJ
and tracking the fluid fronts in each channel. Once split into these three channels, the feeds will be in black and
white, and the intensity of the grayscale pixels can be quantified using ImageJ. This approach may introduce
more sources of error, however, as even in the green channel for example, residues of the red and blue
colors are still visible and may affect intensity measurements.
Phase 2: Integration of Nucleoprotein Assay Reagents Including Label and Capture Antibodies, Wash
Buffers, and Gold Nanoparticles for Detection
Approach
In this phase, the protein reagents and gold nanoparticles will be prepared and integrated into the paper
network developed in Phase 1. The protein to be detected will be recombinant NP with Histidine Tag from
Influenza A/Brisbane/10/2007 (H3N2, Influenza Reagent Resource #FR-480). The lab receives these proteins
12

in a prepared mix (213 g in 250 L of 50 mM Tris-HCl and 600 mM NaCl with 15% glycerol (w/v), pH 8) that
we thaw when preparing the sample for use. The capture antibody will be InA108 monoclonal mouse antiinfluenza virus type A (C/N 3IN5, HyTest Ltd), and the detection antibody will be a biotinylated version of
InA245 monoclonal mouse anti-influenza virus type A (C/N 3IN5B, HyTest Ltd). Both of these antibodies will be
spotted onto the membranes to create detection and control lines. In addition, detection antibodies will be dried
onto glass fiber pads with trehalose, a sugar added to preserve protein shape upon dehydration. The gold
conjugate nanoparticles P/N CGSTV-0600 (Arista Biologicals Inc., Allentown, PA) used for creating a visible
detection line will be dried down onto a glass fiber pad as well. All these dried reagents will be rehydrated by a
buffer solution running through the NC strip consisting of phosphate-buffered saline (PBS, Sigma Packet P3813) with 0.1% Tween-20 (PBST), 4% bovine serum albumin (BSA, # A3059-100G, Sigma-Aldrich, St. Louis,
MO), and 5% Triton X-100. The rationale behind this assayincluding buffer recipe, choice of gold
nanoparticles, and NC HF135 papercomes from results the Yager Lab has seen in previous optimization
tests for this particular NP assay (pending publication). Adjustments to these amounts and the device will be
made to improve the assay performance in the design. The capture antibody will be spotted onto paper
membranes using a SciFLEXARRAYER S3 (Scienion AG, Berlin, Germany) spotting machine. Concentrations
of stock solutions will be taken using a NanoDrop 1000 spectrophotometer and diluted to target concentrations
in PBST to better control and calculate reagent amounts used.

Once the transition to antibodies and gold nanoparticles has been tuned to produce clear detection and control
lines, images of these bands captured by the same webcam set-up as in Phase 1 will be analyzed using a
MATLAB color intensity program. Further changes to the device will be made as necessary until the formation
of colored lines (both detection and control) that are 3x in color intensity compared to background is achieved.
Multiple trials will be conducted to affirm and assess the consistency of results.
Deliverables
The main deliverable of this phase is the creation of a device that successfully integrates a paper network to
perform an unamplified NP assay. Success will be judged by two conditions: a) the production of clear
detection and control colored lines when run with a sample of recombinant NP and b) the presence of only a
colored control line in a trial run in the absence of NP. Data collected will be in the form of multiple videos that
demonstrate the efficacy and consistency of the device.
Anticipated Outcomes
In the testing of the assay, we will have to consider the incorporation of wash steps to remove excess reagents
and improve the signal to noise ratio in our device. Each wash step requires the implementation of another
glass fiber pad, which increases the total time required in the operation of the device, and so ultimately we will
have to consider the trade-off between a longer time required to run the assay and clearer lines. I anticipate
having to conduct a series of experiments with varying wash steps to measure the impact on the formation of
detection and control lines using a quantitative color intensity analysis.
13

Phase 3: Integration of Swab Delivery and Lysis

Approach
The focus of Phase 3 is to develop the interface in which a user would insert a nasal swab with sample to
initiate the device. The swab insertion point on the device will also serve as a reservoir for storage of lysis
buffer, which will have the capacity to lyse viral samples to release NP. This phase will involve using the 3D
CAD package SolidWorks (Dassault Systmes, Vlizy-Villacoublay, France) to draw designs of the swab case.
We will use the labs Objet30 Scholar 3D printer (Tasman Machinery Ltd, Victoria, Australia) to create the case
out of plastic. As previously stated, the rationale for this approach comes from previous work that has been
done in the MAD-NAAT project. However, experience in MAD-NAAT has reported some previous issues with
the compatibility of the 3D printer plastic and the nucleic acid amplification lysis buffer. Whether or not the
plastic from this particular 3D printer will be compatible with the flu reagents is unknown, and so additional
steps yet to be determined may be required to coat or treat the plastic in the later stages of this phase. The
lysis buffer will be the same buffer used in Phase 2. To test the efficacy of lysis, Influenza A virus samples of
various strains (Virapur, San Diego, CA) will be delivered via swab to the device. Once again, we will be taking
webcam videos to analyze the time it takes for the sample to be delivered and the resulting color intensities of
the detection and control lines. Multiple trials will be conducted to affirm and assess the consistency of results.

Note that the swab delivery included in this proposal is limited to the delivery of isolated viral protein and whole
virus harvested from a chicken egg. On a real swab, the viral sample would also be heavily coated in human
nasal matrix. The Yager Lab does have means to create simulant nasal matrix that would be used to mimic a
nasal swab sample. However, due to the fact that the funding source for this nasal matrix is under a grant for
another project, it is not certain I will have access to this in the development of this project. As a result, testing
using simulated nasal samples is not directly included in this proposal.
Deliverables
The main deliverable of this phase is the creation of a swab transfer case that is compatible with the device
crafted in Phase 2. This case will serve as the site of lysis for a viral sample and directly deliver the lysed
solution to the paper network.
Anticipated Outcomes
As already mentioned, one issue that may come up in the development of the swab transfer case is a chemical
incompatibility between the reagents and the 3D printer plastic. Members of the Yager Lab have previously
reported that using a 3D printed sample transfer case significantly decreased the signal to noise output of a
nucleic acid detection test, which suggested that the plastic was negatively impacting the quality of the
reagents. This problem is known but not yet fully understood, and so I anticipate having to do a series of tests
to see if the 3D plastic has an effect on the NP reagents or lysis solution. I am also ready to explore the
possibility of having to coat the 3D plastic in some sort of protective layer or forgo use of the 3D printer and

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manually craft a case from PMMA, a material that we already use in conjunction with reagents, using chemical
solvent welding or standard adhesives.

Engineering Design Standards

Because this project involves manufacturing a device intended for use as a medical diagnostic, this project is
regulated by Quality System (QS) Regulation/Medical Device Good Manufacturing Practices [6]. I will have to
follow the regulations set forth by 21 CFR 820, which regulates that, as a manufacturer, I must ensure that my
device meets a series of safety standards both in the process of its development and in its final usage [7].
Given the short span of my capstone project, I will not be applying for extensive approval from the FDA for my
device to be used in trials with human samples, but I am still required to participate in announced or
unannounced FDA audits should these occur in the year I am working on my capstone. I am to develop a
Quality Management Systems report detailing the potential risks presented by my device, the process involved
in operation and manufacturing of my device, and an overall assessment, including facilities and personnel, of
the Yager Labs ability to manufacture this medical device.

Key Personnel
Aside from Dr. Paul Yager himself, people important to the success of this project include my direct mentor
postdoctoral fellow Shichu Huang, graduate students Koji Abe, Carly Holstein, and Gina Fridley, and
postdoctoral fellow Paula Ladd. Each member of this team has a different area of specialization within the labs
influenza project funded by the National Institutes of Health (NIH) and will be collectively offering ongoing input
and suggestions. In the past, postdoctoral fellow Bhushan Toley served as a direct mentor in my previous work
on switching and valving paper networks, which will become an important part of this capstone project. Other
important personnel for the success of this project include Maxwell Wheeler, one of Yager Labs lab
technicians, and our collaborators from PATH in Seattle, WA, from whom we have adopted and adapted the
nucleoprotein assay I will be using in my project.

As for lab communication, the people involved in the NIH influenza project gather every week for a group
meeting. I will be presenting updates on my project in these meetings and receive feedback in this fashion,
including from Dr. Yager himself. In addition to this, I will be meeting with Dr. Yager individually at least
monthly in the duration of this project for additional mentoring specifically on the progression of my capstone.

Equipment, Facilities, and Resources

The development of this project will take place in laboratory spaces N433C and N533C in the William H. Foege
building, University of Washington Department of Bioengineering in Seattle. The experiments using live virus
samples will be conducted in the Yager Labs biosafety level 2 (BSL2) lab rooms situated in N433C. Equipment
required for this project to succeed include the labs CO2 laser cutter (Universal Laser Systems, Scottsdale,
AZ), spotting machine SciFLEXARRAYER S3 (Scienion AG, Berlin, Germany), and Objet30 Scholar 3D printer
(Tasman Machinery Ltd, Victoria, Australia). Funding for personnel and the resources required in this project
are supported by the labs grant from the National Institutes of Health (NIH NIAID).
15

Figure 5. Engineering design process for portrayed in a flow chart.

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References
[1] Center for Disease Control and Prevention. Reconstruction of the 1918 Influenza Pandemic Virus. 12 May
2014. <http://www.cdc.gov/flu/about/qa/1918flupandemic.htm>.
[2] Center for Disease Control and Prevention. Rapid Diagnostic Testing for Influenza: Information for Clinical
Laboratory Directors. 12 May 2014. <http://www.cdc.gov/flu/professionals/diagnosis/rapidlab.htm#table2>.
[3] Food and Drug Administration. Blood Human Chorionic Gonadotropin (hCG) Assays. 15 May 2014.
<http://www.fda.gov/MedicalDevices/Safety/AlertsandNotices/TipsandArticlesonDeviceSafety/
ucm109390.htm>.
[4] Alere Influenza A&B. 15 May 2014. <http://www.alere.com/us/en/product-details/influenza-a-b-test.html>.
[5] E. Fu, T. Liang, P. Spicar-Mihalic, J. Houghtaling, S. Ramachandran and P. Yager, Anal Chem, 2012, 84,
4574-4579.
[6] U.S. Food and Drug Administration. Quality Systems/Medical Device Good Manufacturing Practices. 23
May 2014. <http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/PostmarketRequirements/
QualitySystemsRegulations/>.
[7] U.S. Food and Drug Administration. CFR Code of Federal Regulations Title 21. 23 May 2014.
<http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=820&showFR=1>.
[8] Center for Disease Control and Prevention. Influenza Type A and Subtypes. 4 June 2014.
<http://www.cdc.gov/flu/avianflu/influenza-a-virus-subtypes.htm>.
[9] Center for Disease Control and Prevention. Influenza Symptoms and the Role of Laboratory Diagnostics.
4 June 2014. < http://www.cdc.gov/flu/professionals/diagnosis/labrolesprocedures.htm#diagnostic>
[10] G. E. Fridley, H. Q. Le, E. Fu and P. Yager, Lab Chip, 2012, 12, 4321-4327.
[11] P. Yager, E. Fu and B. Lutz, Nucleic-Acid-Based Detection of Bacterial Infections Using a Fully-Disposable
Paper-Based Microfluidic Technology, 2013.
[12] Centers for Disease Control and Prevention. Guidance for Clinicians on the Use of Rapid Influenza
Diagnostic Tests. 5 June 2014. < http://www.cdc.gov/flu/professionals/diagnosis/
clinician_guidance_ridt.htm>.
[13] Cepheid. Global Distributors. 5 June 2014. <http://www.cepheid.com/us/about-us/global-distributors>.

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Appendix
I. Request for Proposal (RFP)
Designing Methods to Reduce User Steps in a Flu Protein Detection Module
This proposal responds to a request from the Yager Lab.
The Yager Lab seeks a solution to the creation of affordable, easy-to-use paper diagnostic for the detection of
nucleoprotein (NP) on the inside of the influenza virus that utilizes dynamic paper valving and other mechanical
or chemical systems to allow for activation of the device using a single step.
The Yager Lab requests the following deliverables:
1. A comprehensive evaluation of the automation techniques that can be implemented into the NP device
during three main processes in the device:
a. Preparation of the nasal sample (including soaking the nasal swab in buffer or similar solution
and lysis)
b. Delivery of the nasal sample to the detection region
c. Simultaneous delivery of the detection reagents to the detection region
2. Contribution (from doing the above items) towards accomplishing the labs goal: formulating a complete
blueprint for a device that performs the NP test and meets the following criteria
a. Operates in < 30 minutes
b. Activates in one step that could be done by the lay person using one hand
c. Reproducible, has good sensitivity, gives quantifiable results
d. Requires no refrigeration or electricity
e. Has a shelf life of at least one year
f. Implements controls to confirm the successful transport of reagents and nasal sample to
detection region

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II. Concept Sheet

19

III. Milestones and Timeline Table

Action:
Finalize and test design using
reagent simulations (i.e. dried
down colored sugars in the
place of protein reagents and
colored fluids for buffer and
lysis solutions)

To be completed by:
Ideally by mid-Autumn
quarter next year, target
for by Thanksgiving Break
in November

Criteria for accomplishment:

-Delivers all fluids to the place of hypothetical
detection line
-Integrates introduction of sample, label
antibody, gold nanoparticle labels, and buffer
wash steps
-Demonstrated consistent timing of fluid delivery
-MATLAB color analysis to determine that
colored fluids reach the detection and control
lines in the correct order and at the appropriate
time
Test design using actual
During Winter Quarter,
-Maintains the fluid delivery functionalities
reagents
target for by Spring Break (timing, consistency) that the model with colored
reagents did
-Will show a detection line that is at least 3x the
intensity of the background (MATLAB image
processing)
-Will show a control line that is at least 3x the
intensity of the background (MATLAB image
processing)
-Under repeated trials will consistently express
the appropriate positive and negative test result
Design swab transfer
By end of third week of
-Allows user to insert and twist swab to release
prototype (case for lysis and
Spring Quarter
sample
direct delivery of the sample +
-Stores enough lysis solution to complete lysis
lysis solution to device)
-Delivers fluid onto a paper strip after completion
of lysis step consistently in multiple trials
Incorporate swab transfer
By end of finals week in
-User can complete all steps from insertion of
case with assay device
Spring Quarter
swab to test result
-One (or max two) user steps
-Assay creates detection and control lines as it
did in device without the swab transfer case
-Multiple trials to demonstrate consistency
Table 2. Activities, timeline, and criteria of accomplishment for stages of the project.

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