A Multi-Biomarker Diagnostic for

Cardiac Disease:
Rapid. Portable. Ultra-Low Cost.

Quick Review: Cardiac Disease

Cardiac disease is the leading cause of death in the world; more

so than cancer, respiratory ailments, or HIV [1]
Problem in both developing and developed states
80% of Cardiac disease deaths occur in low-income
countries with poor healthcare infrastructures [4]
Responsible for 1 out of every 3 deaths in the US, more
than all cancers combined [2]

Treatment is costly
Costs healthcare system 444 billion dollars annually or one
out of every 6 dollars spent on healthcare [7]

Like HIV and cancer, early detection of Cardiac disease is

essential so that long-term treatment can begin to improve quality
of life and reduce risk of death
No single, simple treatment
Cardiac disease treatment = long-term lifestyle
management

To manage the disease, patients need accurate multi-biomarker

risk assessment diagnostic that gives feedback on lifestyle
management.

The World Health Organization chart

shown above ranks Cardiac disease
(Ischemic Heart Disease and Stroke) as
the leading causes of death in the world,
above HIV and respiratory infections. [1]

Whats the Problem?

Current standard diagnostic tests target a single biomarker, blood cholesterol

Current biomarker: total blood cholesterol (TBC) and cholesterol within high (HDL-C) and low
density lipoproteins (LDL-C). LDL-C is not measured directly; rather, estimated using other
biomarkers [5, 12]
Cholesterol biomarkers, especially LDL-C and TBC, do not accurately discriminate healthy and
diseased [14,15]
Individuals with both high and low concentrations of current biomarkers may have similar risk of
cardiac disease due to their weak correlation with Cardiac Disease [15]

Current diagnostics are run too infrequently to provide meaningful data to patients
Standard lipid and cholesterol tests run once every 6 months, on average
more frequent testing desirable to assess risks and development of cardiac disease

Current diagnostics for cardiac disease are expensive

Standard lipid and cholesterol test = $50-$100 -Cost too high for families in developing states

Current diagnostics necessitate labs and trained technicians

Developing states with weak health care infrastructures lack both labs and trained technicians

Ox-LDL: A New Biomarker

New, More Accurate Biomarker, Oxidized LDL (OxLDL)
Recent research has discovered a new biomarker of
cardiac disease: Oxidized - LDL that has a far stronger
correlation with cardiac disease than any of the current
biomarkers (see figure on right) [15, 16,17]. This is
because ox-LDL generates an immune response and is
swarmed by masses of immune cells. These immune
cells ingest ox-LDL and become foam cells which form
plaque, the hallmark of cardiac disease. Ox-LDL is
devastatingly potent in its ability to initiate and
accelerate atherosclerosis [16,17], the formation of
plaque in artery walls.

Current Ox-LDL Tests:

HIghly specialized, offered at only a handful of

locations in the US
Cost = $200+ [14]
Far costlier than cholesterol tests ($50+)
Detect oxidization of LDL in last stages, when
irreversible conformational changes occur to the
apolipoprotein- of LDL [14]
By this time ox-LDL is viewed as an antigen
and binds to scavenger receptors of
endothelial cells to create foam cells

Above: The x-axis divides concentrations of biomarkers, from

least to greatest, into 5 groups of patients. The y-axis shows
the percentage of patients within those groups who have
cardiac disease. As a ox-LDL (green) levels increase from each
group to the next, the percentage of patients with cardiac
disease increases greatly; As LDL-C (blue) levels increase, the
percentage of patients with cardiac disease remains roughly
constant. [6] I created the above bar graph using data from
(Johnston, 2006) [Ref. 6]

Ox-LDL:
The Solution
A New Biomarker
To arrive at my solution, I contemplated on the findings of my research; 1) cardiac disease is the

number one cause of death in the world, 2) the cost of diagnostics and the health infrastructure of
poor nations are important factors, 3) traditional diagnostics are based on cholesterol as a biomarker
(especially LDL-C, commonly called bad: cholesterol) 4) whereas its oxidized form more accurately
predicts cardiac disease risk, and 5) current tests are infrequent, lab-based, and expensive.
From a biomarker standpoint, my conceptual solution was a biosensor built around the Ox-LDL
biomarker. This biomarker would keep close track of atherosclerosis initiation and progress caused by
ox-LDL and a cholesterol biomarker to assess the availability of cholesterol contributing to the Ox-LDL
process. From an accessibility standpoint, my solution was driven by cost, portability, and ease of use.
Thus, my research goals were to develop
-

biosensor with multiple biomarkers that accurately reflect cardiac disease state; ox-LDL and
cholesterol
biomarkers to be detected from a single drop of blood
portable, rapid, ultra low cost
scalable fabrication for large-scale production without compromising accuracy or sensitivity

Research Overview
Ox-LDL Sensor
Two detection methods: Colorimetric and Electrochemical
Each method has its own merits: the colorimetric test is
ultra-low cost and the electrochemical test allows for
more quantitative metrics.
OX-LDL Colorimetric Sensor:
The iodometric reaction was shown by El-Saadani et al. to
detect lipid peroxides (ROOH) of Ox-LDL [21]. The reaction
(see below) produces the yellow triiodide ion. This works
reliance on incubation, spectrophotometric absorption
analysis, and large sample volume, make it a lab-only test.
Iodometric detection using a portable paper-based sensor,
to detect ox-LDL has not been reported in published works.

Ox-LDL Electrochemical Sensor:

During early stages of oxidation, lipid peroxides form on oxLDL [20]. In functionalized carbon nanotubes (fCNTs),
hydroxyl and carboxyl groups enable electronegative
interactions that detect the presence of lipid peroxides.
Furthermore, carbon nanotubes have a natural affinity
towards lipids.

Cholesterol Sensor
The main goal was to develop a
paper-based sensing platform [18,
22, 23] that could be produced
cheaply with a colorimetric test
that reacts to the blood
cholesterol with a color change, is
captured by a smart phone, and
analysed with software. Paper
substrates have been researched
for various analytes for color as
well as electrochemical detection.
This work is specifically for the
cholesterol biomarker using a
consumer grade modified inkjet
printer (< $50) for printing
enzymatic and dye reagent ink on
cellulosic paper.
Color change is detected when the reaction below occurs
in the presence of enzymes and a chromogenic dye.
Enzyme inks with three such dyes were tested, 4-AAP,
TMB, and ADHP. The ink was deposited upon cellulosic
substrate using consumer inkjet printing technology.

Year 1: Electrochemical Ox-LDL Sensor

Year 1 TESTS: The sensor requires a 3-electrode system and
electronics for measuring currents elicited by electrochemical
charge transfers. For measurement electronics, a commercial
blood glucose meter was hacked and the sensor electrode leads
connected directly to the posts of the glucose meter. The counter
electrode was made with a Pt wire with the end coiled. A Ag/AgCl
reference electrode was made by dipping in ferric chloride..

Functionalizing Carbon Nanotubes, Making Working

Electrode:
The CNTs were functionalized by immersion in an acid bath of nitric
acid, sulfuric acid, and hydrogen peroxide for 5 hours. After
filtration and several washes in distilled water, the CNTs were dried
(300C) for 2 hours. A transfer pipette with Cu wire was evenly
coated with epoxy/graphite mixture, rolled in the functionalized
CNTs, and the exposed ends of the copper wire tightly wrapped
around.

Preparing Oxidized-LDL Test Sample

Native LDL was purchased from Sigma Aldrich and oxidized in
CuSO4. 10 mL of a 5 M solution of CuSO4 was prepared to which
75 L of native-LDL (40 mg/ml) solution was added. The resultant
0.3 mg/ml LDL/ CuSO4 solution was incubated in a water bath at 37
C. This work was performed in the biosafety lab of BUGSS. The
sensor was tested by placing the 3 electrodes in an ELISA plate well
containing 30 l of Ox-LDL and initiating the glucose meter.

In the early stages of oxidation, lipid peroxides are formed. The

electrochemical sensor developed here is based on detecting lipid
peroxides and the increased electronegativity of
Ox-LDL.
Accordingly, functionalized carbon nanotubes, that have hydroxyl
and carboxyl groups sensitive to lipid peroxides, form the basis of
Ox-LDL detection.

Year 2: Electrochemical Ox-LDL Sensor

Year 1 TESTS = FAILURE
Year 1 tests with the hacked blood glucose meter gave an error signal, Err for all Ox-LDL test samples. Tests performed
with H2O2 instead of Ox-LDL, gave the code LOW, indicating that even in high concentrations of H 2O2 the levels were
below what the meter expected. With failure experienced with the Phase 1 tests, I read books and research publications to
teach myself electrochemistry. In my readings, I found out that a potentiostat would enable me to design my experiment to
measure the small currents produces, and read of the cheapstat potentiostat that costs under $100. I wrote to Dr. Ryan
White, a researcher that worked with this potentiostat, who then provided me with a free cheapstat.

Year 2 TESTS:
Year 2 tests were conducted with the open-source Cheapstat potentiostat. The firmware of the
potentiostat was installed and a Java Runtime executable was activated for measurement.

fCNT 3-electrode assembly

fCNT 3-electrode assembly in solution

Au electrode in 3-electrode assembly

Cheapstat Potentiostat

In addition to the fCNT working electrode of the Phase 1 tests, two other electrodes were also tested. The ability of three
electrode materials, gold, graphite and fCNT, to detect various concentrations of hydrogen peroxide test solutions, were
performed. Potentiostat measurements for each test was obtained with cyclic (CV) and linear sweep (LSV) voltammetry.
Because the functionalized carbon nanotube working electrode detected H2O2 at very low concentrations, the fCNT
electrode was then used in tests with native and oxidized LDL.

Year 2: Colorimetric Ox-LDL Sensor

To create my sensor, I prepared a sheet of Whatman Grade 1 qualitative

filter paper:
Reagents potassium phosphate (0.4M) and potassium iodide (0.24M)
were pipetted onto the cellulosic substrate
Since my sensor worked by detecting hydroperoxides, and I had a limited
quantity of LDL samples, I first tested the sensor with hydrogen peroxide, a
hydroperoxide, to test my principle of detection
This would confirm whether the sensor could detect the lipid
peroxides of ox-LDL

Once the color of the sensor had fully developed, I took a picture with
a smartphone camera (iPhone 5) to ensure the test could be
performed in-homes and health clinics
I tested at varying concentrations of peroxide to determine if there
might be sufficient color differentiation
I ran the tests several times to ensure reproducibility of results
After testing the sensor with hydrogen peroxide, 1 M to 400 M, I then
tested the sensor with actual ox-LDL samples
Native LDL was oxidized as described in the preceding slide and OxLDL samples Ox-LDL samples were then tested at the 0, 1, and 2 hrs
intervals.

(Left and right) working at University of Marylands Bioengineering Lab to fabricate my colorimetric ox-LDL
sensor

Year 1: Cholesterol Sensor

Year 1 Work

I first ensured that the chemistry of my sensor was reliable and

accurate before inkjet printing the sensor. For this, I pipetted the
mixture of enzymes, dye, and different cholesterol concentrations
onto the paper-sensor.
Once the color had fully developed, I took a picture with a
smartphone camera (iPhone 5) - I wanted the test to have ease of
use and accuracy for use in field based settings.
Tests were repeated to ensure consistent results and to find
optimal dye/enzyme concentration.
The smartphone image was analyzed using image software
ImageJ
Image analysis indicated sensor accuracy and significant color
differentiation between cholesterol concentrations

Fabricating and testing sensors at the

Baltimore Underground Science Space, a
community hackerspace, where I conducted
phase one research.

Cholesterol, Dye Solution

Cholesterol was mixed in detergent TritonX100, and then in 10 mM phosphate buffer solution (PBS) to yield
cholesterol solutions of 50, 100, 150, 200, 250, 300, 400, 500 mg/dl. The dye tetramethylbenzidine (TMB) was mixed
with DMSO (5.8 mg/ml).
Enzyme, Dye, Cholesterol Test Solution
A test sample of 15 L of cholesterol solution was pipetted into a well of a 96-well plate, followed by 5 L each of
enzyme cholesterol oxidase (100 IU/ml), horseradish peroxidase (25IU/ml), and dye TMB. The enzyme solutions were
prepared in 10 mM PBS. The entire solution was then transferred to a chromatography paper strip (2 cm x 0.7 cm).

Year 2: Cholesterol Sensor

Year 2 Work

Once I had proven the chemistry of my sensor to be reliable and accurate I moved on to create a fabrication method
that was both ultra-low cost and scalable to mass production, inkjet printing [23].
I used an inexpensive consumer inkjet printer ($30) to accomplish both objectives
I retrofitted a consumer inkjet printer to print the enzymatic inks I wanted
I removed the printers covering assembly exposing the cartridges, rails, and electronics below, and secured
shut the paper clamp to trick the printer into printing my test strips
I used surgical tubing to charge my ink, increased the cartridges air pressure to print smaller volumes, created
pseudo-cartridges to overcome the proprietary protections and modified ink cartridges to print my ink
Ten ink recipes were engineered to optimize flow dynamics and avoid recurrent problems with ink dripping and
nozzle clogging while maximizing enzymatic sensitivity.
To my ink I added: Triton-x-100, usually used to lyse cells, which I repurposed to eliminate bubbles that
caused ink dripping and glycerol to increase the viscosity of ink and ensure an accurate print.
I tested two printing systems: piezoelectric Epson Workforce 30 and thermal HP Deskjet 1010.
After hundreds of failed attempts, dozens of clogged cartridges, and three destroyed Epson printers, I decided
to use the HP printer instead.
To ensure printing functionality, a blank ink (glycerol and H2O) with fluorescent tracer Fluorescein was tested.
Having demonstrated viability, I printed my enzymatic inks with one of three dyes: Tetramethylbenzidine (TMB), 4Aminoantipyrine (4-AAP) or 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP).
I printed three different dyes to determine which would provide the greatest color differentiation.
The inks were printed onto Whatman Grade 1 qualitative analysis paper.
Resultant test strips were tested with known concentrations of cholesterol, and once the color had fully
developed an image was taken on a smartphone for later analysis

Year 2: Cholesterol Sensor (cont.)

Retrofitted piezoelectric
Epson Workforce 30
(top right) and thermal HP
Deskjet 1010 (top left.)
Me working at University
of Marylands
Bioengineering Research
Lab (bottom left.) Cleaned
HP Deskjet 1010 Ink
Cartridge (bottom right)

Results: Ox-LDL Electrochemical Sensor

Gold working electrode performance

Graphite working electrode performance

FCNT working electrode performance

The above graphs compare the LSV values of the fCNT electrode (top
right) against gold (top left) and graphite (top center). The fCNT electrode
was routinely able to detect peroxides (H2O2) at very low concentrations
of 0.22M, whereas its graphite and gold counterparts were only able to
detect peroxides in the upper mM range. The FCNT detectors high
sensitivity to ultra-low concentrations of peroxides demonstrates its
potential for detecting lipid peroxides in ox-LDL from blood samples,
which are commonly found in low M concentrations [13].
The functionalized carbon nanotube (fCNT) sensor response (right) to
native LDL (0 hours) and oxidized LDL (1 hour and 2 hours oxidation
time) shows increasing current measurements as the oxidization of
LDL progresses. Thus, confirming that the fCNT sensor can indeed
detect Ox-LDL in concentrations found in blood.

Graph of current detected vs. oxidation time of native

LDL in hours showing

Functionalized carbon nanotube sensor

performance with ox-LDL

Results: Ox-LDL Colorimetric Sensor

The results for the iodometric sensor response to varying
concentrations of hydrogen peroxides are seen in the figure to the left.
Visual differentiability clearly exists between high concentrations
greater than 300 M and low concentrations of 100 M. In the 1-200
M range, sufficient differentiability between concentrations was not
present. to warrant RGB histogram image analysis. However, it is
thought that more subtle changes in color may be quantified with a hue
and saturation value image analysis, which will be investigated in future
work.
Right: Color differentiation for hydrogen peroxides samples

The figure to the right shows the sensor response to

progression of LDLs oxidation over time. It is seen that as
oxidation progressed, the sensor responded with increasing
color intensity. This change in color intensity was more
apparent at the boundaries of the sensor. As with the hydrogen
peroxide test samples, color differentiation was not great
enough to warrant RGB image analysis. However, the sensor
can alert users to high-low concentrations of ox-LDL. Though
less sensitive to ox-LDL than the electrochemical sensor, the
colorimetric sensor is extremely inexpensive; the cost of the
sensor equals the cost of the paper substrate due to the ultra

Above: Color differentiation for oxidized-LDL samples

Results: Cholesterol Sensor

Metrics of color change against cholesterol concentration are shown below. Histogram analysis was performed on the
sensors circular reaction zone ROI using ImageJ. A linear trend line is observed for cholesterol concentrations 100400 mg/dL for all dyes, demonstrating a linear model for relating color intensity to cholesterol concentration (R 2 >
0.9). Thus, image histogram analysis can provide a quantitative measure of the cholesterol concentrations present.

The sum of histogram mean intensities of the

background corrected blue and green RGB
channels for the TMB dye sensor vs.
cholesterol concentration (100-400 mg/dL)

The difference between histogram mean

intensities of the red and RGB channels for the
4-AAP dye sensor vs. cholesterol concentration
(100-400 mg/dL)

The difference between histogram mean

intensities of the red and RGB channels for the
ADHP dye sensor vs. cholesterol concentration
(100-400 mg/dL)

TMB: The green-blue color change observed for TMB occurred rapidly, within 30 seconds of sample deposition.
However, with higher concentrations (>300mg/dL) double oxidation occurred, producing yellowed hues.

4-AAP: The orange hued color change observed for 4-AAP occurred, out of all three dyes, the slowest, taking about 6
minutes for full color development after sample deposition. However, it did yield homogenous color distribution, exhibits
greater differentiation in the lower ranges of cholesterol, and does not double oxidize.
ADHP: The magenta change is slower than TMB, but quicker than 4-AAP. That secondary color changing oxidations do
not occur, unlike TMB, is an advantage. It also exhibits vibrant color differentiation well suited for high concentrations.

Conclusions
For the past two years, through failures and successes alike, Ive strived to meet my research goal. Heres what I
conclude:

Ox-LDL Sensors

Inkjet-Printed Cholesterol Sensor

fCNT sensor routinely detected peroxide samples as low as

0.22 M, accurate enough to detect the lowest concentrations
of ox-LDL in blood. The fCNT working electrode outperformed its
graphite and gold counterparts several times over, thus proving
the principle of detecting hydroperoxides with with fCNTs.

Successfully provided quantifiable color

differentiation accurate for field use, with a linear
relationship established between cholesterol
concentrations and color intensity across three
discrete chromogenic dyes.

The functionalized carbon nanotube electrochemical sensor

successfully detected ox-LDL at concentrations comparable to
that of human blood and was able to establish a linear
correlation between ox-LDL concentration and detected current.

Ultra-low cost: 2 cents per strip.

The iodometric ox-LDL sensor provided clear color

differentiability to the naked eye for low/high concentrations of
hydrogen peroxide proving that peroxides can be detected on
paper sensors with the iodometric method.
The tests with Ox-LDL provided limited color differentiability,
but still visible for differentiating very low and high ox-LDL
concentrations. This novel method is the first instance of using
a paper-based sensor coupled with the iodometric method for
detection of Ox-LDL.
Both sensors detect ox-LDL in the earliest stages of its
oxidation whereas traditional ELISA tests detect ox-LDL in its
last stage of oxidation.

Fraction of home and lab-based tests; only made

possible by the novel micro-scale enzyme
deposition printing system employed. The inkjet
printing systems scalability presents further
opportunities to lower marginal costs.

Problems and Interferences

The cholesterol Sensor has specificity to the
cholesterol biomarker with the use of cholesterol
oxidase enzyme eliminating problems of interference.
Interference can occur for the Ox-LDL sensor from
other non-lipid hydroperoxides.Extremely small
interferences from H2O2 can be eliminated with
enzyme Catalase which will reduce it. Protein
peroxides can be corrected with the use of
triphenylphosphine (TPP) which will reduce the
hydroperoxides to alcohol.

Marketability Evaluation
Costs

My on-site Ox-LDL Electrochemical Sensors projected cost $20

- Current Lab-Based ELISA Test > $200+
- Sensor will be reusable in final design with fCNTs electrodes: much like glucose meter electronics
My on-site Ox-LDL Colorimetric Sensors Cost < $0.01
- No current paper-based colorimetric Ox-LDL tests available up until this research
My on-site Cholesterol Sensors Cost projected $0.02
- About 200 times cheaper than current home-tests
- Over 2,000 times cheaper than current lab-test
- Ultra-low cost made possible by the inkjet printing system used in this research. The printer
precisely places each droplet of ink, greatly conserving the use of expensive enzyme ink.
Traditional less efficient fabrication methods (eg: dip coating, screen printing) use more reagents.

Time from Sample to Answer

My on-site Ox-LDL Electrochemical Sensor (avg.) 35 seconds

-Current lab-based ELISA Test = 3-4 weeks
My on-site Ox-LDL Colorimetric Sensor (avg.) 30 minutes
My on-site Cholesterol Sensor (avg.) 30 seconds (TMB dye), 60 seconds (ADHP dye), 6 minutes (4-AAP
dye)
-Current lab-based Cholesterol Test = 1-3 weeks

Final Remarks

With further refinements, patients can receive their ox-LDL levels in less than 30 seconds with the
speed and accuracy of the electrochemical fCNT sensor.
This sensor has the power to transform how we think about cardiac disease from one attributed to
bad and good cholesterol to a disease of oxidation and inflammation.
The speed and ease of use of the sensor allows for non-specialists in home settings to receive
immediate health feedback. Although the iodometric color sensor provides less sensitivity for ox-LDL,
its simplicity and ultra-low cost enhances its utility.

Future Work
The inkjet printing system developed here replace traditional, more expensive paper microfluidic fabrication methods. It can cheaply
mass-produce diagnostics tests for ABO antigens, pathogenic bacteria marker proteins, and more in addition to environmental
monitoring and food purity tests. I envision a home consumer inkjet printer that uses pre-made and packaged reagent cartridges to
fabricate any diagnostic test needed from a pregnancy to flu tests. This printer can rapidly provide vital healthcare statistics to
doctors and patients, sowing the seeds of a personalized medicine revolution that allows for earlier detection of diseases and
treatment tailored to the physiological response and conditions of the patient.
The work continues to integrate multiple sensors into a single holistic unit
(See Future Lab-on-Paper Device on left):

A lectin-treated filter removes red blood cells, and the resultant blood
plasma travels up channels into reaction zones

HDL is separated from LDL using dextran sulfate and Mg2+ to

calculate HDL-C levels. HDL is included in the future lab-on-paper unit
because, current research indicates the HDL-C to Ox-LDL ratio is the
best biomarker of cardiac disease [6]. This is because the two
biomarkers are antagonistic: HDL is antioxidative and can slow or
reverse LDLs oxidation [8]

Cholesterol esterase added to the HDL-C and TBC sensor to catalyse

breakdown of cholesterol esters into free cholesterol [5]

Inkjet print hydrophobic barriers (ex: methylsilsesquioxane (MSQ)) to

form microfluidic channels and control sample flow

Inkjet print silver colloid ink circuits to enable the electrochemical

detection of cholesterol via voltammetric oxidation of H2O2

Investigate paper substrates, such as nitrocellulose, for improved

enzyme immobilization and flow dynamics

Investigate hue and saturation values image analysis; more precise

than RGB histogram analysis. This may enable the color quantification
of the ox-LDL iodometric sensor.

Add a weak acid to potassium iodide to increase the prevalence of I 3and increase the ox-LDLs colorimetric sensors sensitivity.

References and Acknowledgements

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Acknowledgements
Much thanks to Dr. Ian White at the University of Maryland (UMD) for providing me access to his lab. After cold emailing over 64 researchers at local
universities, Dr. White graciously agreed to provide me with the space and basic lab equipment needed to do my work, even though our areas of research had
little overlap. More information on Dr. Whites research can be found here: http://goo.gl/6wb61K. Much thanks as well goes to Dr. Whites Ph.d candidates,
Steven, Shawn and John, for their support. Thanks also to Dr. Ryan White, UMD-BC,, for providing me with a CheapStat potentiostat and the Baltimore
Underground Space Center for providing me lab space for the first phase of research. Dr. Karen Swanchara, my high school biology teacher, thank you for
getting me excited about science research as a freshman, and your continued belief in me over the last three years. Finally, thanks mom and dad for driving
me to and from the University of Maryland every weekend through snowstorms and heavy traffic during the hour long commute.