83% of the US workforce faced chronic stress in 2018. Cortisol is the hormone that regulates stress levels in the body. The document describes the development of an inexpensive, easy-to-use lateral flow test strip to accurately measure cortisol levels from urine samples in under 15 minutes, achieving over 96% accuracy. This provides a lower-cost alternative to existing cortisol tests that are more expensive and time-consuming.

2. In 2018, 83% of the United States workplace population faced “chronic” stress.
Although many people know what stress is and what causes stress, very few
people know that cortisol is the hormone in our body that regulates our stress
levels.

3. • Cortisol is a steroid hormone that
is formed in the adrenal cortex,
the outermost part of the adrenal
gland.
Cortisol- Biochemical Stress Marker
• Cortisol runs on a circadian
rhythm, meaning levels fluctuate
throughout the rest of the day.
• Typically, levels are highest at 8
in the morning.
Urine Cortisol
Adrenal Cortex

4. Cortisol- Biochemical Stress Marker
• Cortisol is a regulator of your Allostatic Load,
meaning it controls our parasympathetic
nervous system, our ability to breath
automatically, and our heartbeat
• Cushing’s Syndrome is caused by too much
cortisol.
• Addison’s Disease is caused by too little cortisol.
• Both can result in severe health problems, such
as extreme fluctuations in blood pressure or
blood sugar, or extreme anxiety or depression.

5. • The HPA Axis is a process responsible for over 30%
of our body’s hormone regulation.
• Hypothalamus uses cortisol to produce- CRH,
Dopamine, Oxytocin, etc.
• Pituitary Gland uses CRH to produce- ACTH,
Growth, Thyroid Simulators, etc.
• Adrenal Gland uses ACTH to produce- Cortisol,
Aldosterone, Adrenaline, etc.
• Cortisol = driving factor of HPA Axis, stimulates CRH
production, which stimulates ACTH production,
which completes the cycle.
• Cortisol controls the entire cycle, since it is the only
hormone that is influenced by outside factors.

6. • Few tests already exist for measuring cortisol:
• Synthase Blood Test- uses blood sample
• Laboratory Test- uses urine/saliva sample
• Expensive- anywhere from $80-$400
• Time Consuming- 2-3 weeks to get results.
• Engineering goal was to create a cortisol test
that was:
• Inexpensive
• Easy to use
• Able to provide results rapidly
• Lateral Flow Immunoassay Sample Lateral Flow
Immunoassay- Pregnancy Test

7. Lateral Flow Immunoassay
Conjugate Pad-
Separates urine from cortisol and
binds cortisol to its antibody so it
can form antibody test lines. Backing Card-
Maintains stability
Sample Pad-
Absorbs urine
onto test strip
Absorbent Pad-
Absorbs excess
urine
Antibody Test Lines-
show up depending on how
much cortisol is present
Plastic Cassette- Protects test strip from damage

8. Step 1: Ag-Ab Complex
Step 2: Qualitative Results
Step 3: Neutralization
• Antibody and Antigen (cortisol)
noncovalent bonds and
electrostatic interactions
• Component Activators cause a
qualitative change
• LFIA- when bound with gold
nanoparticles a color change
occurs and lines appear• Immune Complex at Epitope
• Component Activation
• Release of Component Activators
• Antigen cannot be used again
• Antibody and Antigen cannot be
separated.
• These are steps explaining how
cortisol binds to its antibody.

9. Sample Pad and Fiber Pad Selection
• This procedure tested two different glass fibers, 0.48 mm and 0.83
mm, to determine which would absorb the most sample while also
retaining a uniform amount each time.
• This was conducted by dipping the strips into samples of water to
find which absorbed the most.
Glass Fibers
Cellulose
Fiber
.83 mm
.48 mm

10. Sample Pad and Fiber Pad Selection
• 0.83 mm glass fiber retained more than the 0.48 mm glass fiber.
• Standard deviation was smaller for the thicker fiber, meaning uniform
retention was higher.

11. Creation of a Conjugate Pad
• Solution must have a pH between 5.2 and 5.4 (isoelectric point of cortisol)
• Test solution with a PBS and Sodium Salt Gradient
• Should change from gold to purple
• Mixture of PBS and Sodium Salt activates the
component activators and ensures the solution
works without adding cortisol.

12. Enhancement of Antibody and Fluorescent Dyes
• Mixed antibody with Crystal Violet and Fluorescent Dye to make lines.
• Stamped onto test strips
Production of Finalized Test Strip
• All parts were combined and added onto one strip
• Plastic Adhesive added to back

13. Construction of a Plastic Cassette Using a
3D Printer
• TinkerCAD
• Design one was altered to create a
slimmer second design
• Lower Cost
• Black Light glued to top to see if it
had any affect on the line
pigmentation.

14. Design 1 Design 2
Black Lights
Dimensions: 25cm x 3cm x 2cm Dimensions: 20cm x 3cm x 1cm
3D Plastic Cassette Design and Alterations

15. Creation of Synthetic Urine
• Low, Medium, and High Cortisol Amounts
(0.4 uG – 1 uG) were added to different
synthetic urine.
• Amounts were based on typical 8 AM
Cortisol Levels
• Sample Calculations from uG to uL
𝐷𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑐𝑜𝑟𝑡𝑖𝑠𝑜𝑙 = 1.3 ൗ
𝑔
𝑐𝑚3
𝑉𝑜𝑙𝑢𝑚𝑒 =
𝑚𝑎𝑠𝑠
𝑑𝑒𝑛𝑠𝑖𝑡𝑦
𝑪𝒂𝒍𝒄𝒖𝒍𝒂𝒕𝒊𝒐𝒏 𝒐𝒏𝒆: 0.4 𝑢𝐺 𝑐𝑜𝑟𝑡𝑖𝑠𝑜𝑙 = 0.000004 𝑔 𝑐𝑜𝑟𝑡𝑖𝑠𝑜𝑙
𝑉𝑜𝑙𝑢𝑚𝑒 =
𝑚𝑎𝑠𝑠
𝑑𝑒𝑛𝑠𝑖𝑡𝑦
=
0.000004 𝑔
1.3 ൗ
𝑔
𝑐𝑚3
𝑉𝑜𝑙𝑢𝑚𝑒 = 0.0000031 𝑚𝐿 𝑐𝑜𝑟𝑡𝑖𝑠𝑜𝑙 = 0.0031 𝑢𝐿 𝑐𝑜𝑟𝑡𝑖𝑠𝑜𝑙
𝑓𝑜𝑟 20 𝑚𝐿 𝑜𝑓 𝑢𝑟𝑖𝑛𝑒 𝑎𝑑𝑑 0.062 𝑢𝐿 𝑐𝑜𝑟𝑡𝑖𝑠𝑜𝑙

16. 0 Lines 1 Line 2 Lines
• Out of 28 Trials- 27 were
accurate.
• 96% Accuracy
• Trial 19 was inaccurate due
to human error
Accuracy Testing
• 0 lines = too little
• 1 line = within normal
range
• 2 lines = too much

17. • Engineering Goal was completed
• Test has a 96% accuracy
• Standard accepted accuracy is
90%-95%
• Total Cost is $2.03
• Typical cost is $80-$400
• Future Considerations
• Alter design for other hormones
• Conduct more trials
• Find cheaper materials to bring
down the total cost

18. • Pablo Abellán-Galiana, Carmen Fajardo-Montañana, Pedro Riesgo-Suárez, Marcelino PérezBermejo,
Celia Ríos-Pérez, & José Gómez-Vela. (2019). Prognostic usefulness of ACTH in the postoperative
period of Cushing’s disease. Endocrine Connections, (9), 1262.
• Pofi, R., Feliciano, C., Sbardella, E., Argese, N., Woods, C. P., Grossman, A. B., … Tomlinson, J. W. (2018).
The Short Synacthen (Corticotropin) Test Can Be Used to Predict Recovery of Hypothalamo-Pituitary-
Adrenal Axis Function. The Journal of Clinical Endocrinology and Metabolism, 103(8), 3050–3059.
https://doi-org.ezproxy.point.edu/10.1210/jc.2018-00529
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