Ukweli Project UTI Test Strip: Elevated pH Test

Group 5: Haley Janowitz, Madeline Nyblade, Kris Verlinde, Joseph Warner

April 17, 2015

Introduction:
Urinary Tract infections (UTI) are an infection of the urinary tract in which
microbes (mostly bacteria) infiltrate the bladder and/or kidneys. Normally, the body
quickly removes the bacteria that enter into the urinary tract, but some remain and can
cause infections. Urinary tract infections are the second most common infection and most
are not serious, though some can lead to more serious kidney infections (1). Symptoms
vary greatly per patient, but include pain during urination, fever (if the infection has
reached the kidneys), nausea, and vomiting (1). In developing countries, UTIs pose a
large social and economic problem because they are frequently not diagnosed quickly,
increase the risk of contracting HIV or other STIs, potentially lead to severe kidney
damage and complications at birth, and have high occurrence rates (up to 30 - 50% in
pregnant women in sub-Saharan Africa). In order to provide a simple, cost-effective
method for testing UTIs in the field where few doctors are present, the Ukweli project
aims to produce ink-jet test strips to test key features of urine that could indicate a UTI
(2). This report specifically looks at the effectiveness of the pH indicator neutral red
buffered with potassium dihydrogen phosphate/disodium hydrogen phosphate at
detecting abnormal basicity in urine that could potentially indicate a UTI.
The pH of urine can range from 4.6 to 8.0 with an average around 6.0. In a
healthy individual, the kidneys work to maintain the bloods pH at 7.4 by filtering out
substances and excreting them from the body through urine. Therefore, the pH of urine is
variable depending on diet and products of metabolism. If there is more acid in the blood,
the urine will be more acidic in order to compensate and if there is more base in the
blood, the urine will be more basic. Because the pH of urine fluctuates, urine pH cannot
be the only information used to diagnose a patient with any problem (3). This is why pH
is not the only indicator used to determine the presence of a UTI.
Even though urine pH normally fluctuates, abnormally basic urine can be an
indication of a UTI. The bacteria Proteus is responsible for 5 to 10% of all UTIs, among
other Gram negative bacilli bacteria, and produces the enzyme urease that breaks down
urea into ammonia and carbon dioxide (4):
(NH2)2CO + H2O CO2 + 2NH3
Ammonia is a weak base because it can react with water to create ammonium and
hydroxyl groups, raising the pH of the urine and decreasing the pOH:
NH3(aq) + H2O(l) <--> NH4OH(aq) NH4+(aq) + OH-(aq)
Therefore testing for the pH of the urine will likely detect the presence of this enzyme,
which would indicate the presence of a UTI.

The ink-jet test strip proposed by the Ukweli project uses the neutral red indicator
as the pH indicator. At a pH lower than 6.8 the neutral red solution or test strip would be
red, but once the pH rises beyond that point it begins to change orange. At a pH of 8, the
solution or test strip begins to turn yellow (5). The excess hydrogen ions in more acidic
pH (<6.8) solutions bind to the neutral red molecule to form a structure that absorbs green
light and therefore appears red. In a solution with a pH of 8.0 or higher, there are less
hydrogen ions present in solution so the neutral red molecule does not bind with them,
changing its structure so it absorbs violet light and therefore appears yellow. In between
the pH of 6.8 and 8.0 the solution will appear orange because some neutral red molecules
will have hydrogen atoms bonded with their nitrogen and some wont, making a mix of
red and yellow molecules (6).

Figure 1: Neutral Red Reaction (6)

Because the color change happens at a pH of 8.0, the same threshold for the pH of normal
urine, it is a good indicator for abnormally basic urine and the presence of a UTI.
Sensitivity of Neutral Red:
It is assumed that the color change will be visible/ easily identifiable after 80-90%
of the indicator molecules have reacted. Neutral red is the indicator molecule and changes
color from red to orange at a pH of 6.8 and from orange to yellow at a pH of 8.0 (5).
Therefore, the color change from red to orange happens at a pOH of 7.2 and from orange
to yellow at pOH of 6.
Table 1: Hydronium and hydroxide concentration for neutral red color change
Color Change

pH

[H3O+]

pOH

[OH_]

Red to Orange

6.8

10-6.8 mol/L

7.2

10-7.2 mol/L

Orange to Red

8.0

10-8.0 mol/L

6.0

10-6.0 mol/L

pH+pOH = 14: 14 - 6.8 = 7.2

The concentration of OH_ must be greater than 10-6.0 mol/L to change neutral red
completely to yellow and must be greater than 10-7.2 mol/L to start the color change from
red to orange. The normal pH of urine ranges from 4.6 - 8.0 (3), but in the presence of a
UTI caused by Proteus the pH of urine increases due to the increase in OH- ions. If
roughly five microliters of urine is used for each sample, then at least 5x10-12 moles of

OH- ions must be present in the sample for the indicator to change to yellow ((5x106L)
(10-6mol/1L) = 5x1012mol).
The neutral red solution on the test strip is also buffered with potassium
dihydrogen phosphate and disodium hydrogen phosphate, which are effective in
maintaining a pH of 6 to 8 (7). The purpose of these buffers is to prevent any change in
pH, and therefore a color change, from occurring before the actual test is taken. The
buffer keeps the strips acidic so that results cannot be falsified by accidental exposure to
weak bases. This ensures that the accuracy of the test strips is as high as possible. The
buffers are not strong enough to interfere with identifying a UTI since the pH associated
with the medical condition (above a pH of 8.0) is out of the buffers range of
effectiveness (pH 6-8) (7).
Furthermore, this is an acid-base reaction and therefore progresses at a rapid rate.
An almost instantaneous color change in neutral red will be seen if the urine is above a
pH of 8.0 (8). This is useful because test strips can be read immediately to determine the
urines pH, and therefore they will not be exposed to additional contaminants that could
potentially interfere with the pH reading.
Potential Contaminants:
Carbon dioxide has the potential to affect the test strip readings with its presence
in the environment. When carbon dioxide levels increase in the atmosphere, or when
water is left out in the open for an extended period of time, carbon dioxide and water
have the ability to form carbonic acid. Therefore, it could decrease the pH of the sample,
possibly making an abnormally basic pH appear to be within the normal range. This
effect could lead to incorrect test results if the urine was exposed at length to the carbon
dioxide in the air. Therefore, the sample should be tested immediately after being
collected and the test strip should be read immediately.
Other Factors Affecting pH:
Table 2: Components of urine
Urine
Component

Normal Urine (9)

Possible Contents of UTI Urine

(Proteus Infection)

Water

95%

95%

Urea

9.3 - 23.3 g/L

generally less than 9.3 g/L

Creatinine

0.670 -2.15 g/L

Sodium

1.17 - 4.39 g/L

Chloride

1.87 - 8.4 g/L

Potassium

0.750 - 2.61 g/L

Ammonia

.2 - .73 g/L

Ketones

.01 - .05 g/L

Bacteria

Less than 100,000 colonyforming units [CFUs]/ml (10)

greater than .73 g/L

Greater than 100,000 colonyforming units [CFUs]/mL (10)

Some of the dissolved metal ions in the urine have the potential to increase the
acidity if they are present in high concentrations. Metal ions behave like Lewis acids in
aqueous solutions because it attracts the electrons causing H+ ions to be released from the
surrounding water molecules. Sodium, potassium, magnesium and calcium are the ions
typically present and all of them have relatively low charge to size ratios meaning that
they are not extremely acidic and act like spectator ions. As long as they are not present
in abnormal concentrations, then they probably wont affect the readings on the test strips
(8).
Additionally, creatinine and uric acid have the potential to alter the pH of urine if
they are not present in their normal amounts. Abnormal levels can be an indication of a
more serious medical condition and not the result of a UTI. In these cases, these
individuals should already be seeking medical care for their conditions. These conditions
include cancers that have spread, diseases that break down muscle fibers
(rhabdomyolysis) or affect bone marrow (myeloproliferative disorder), gout, kidney
failure and several others (11).
Overall, as long as the components of urine remain at their normal levels,
meaning the patient is healthy aside from the possible UTI, then the pH test strips should
work accurately. If the patient has another existing problem, the pH test may not be an
accurate indication of a UTI. If the patient has a disease, which increases the acidity of
their urine, the basicity of the possible UTI could potentially be masked by acidic
components caused by the other disease. If the patient has a disease, which decreases the
acidity of their urine, the test may come back positive for a UTI though none may be
present. Therefore, pH should not be the only indicator used to determine the presence of
a UTI.
Conclusion:
The neutral red indicator buffered with potassium dihydrogen phosphate and
disodium hydrogen phosphate will indicate if the pH of the urine is above 8.0. This pH is
abnormally basic, and is one indication of a UTI. Because there are other factors that
could alter the pH of urine, other tests should also be made to conclude the presence of a
UTI such as tests for nitrates and leukocytes. The neutral red solution will react with the
urine almost instantaneously, changing the solution yellow if over a pH of 8.0, so a quick

reading will be possible. It is best to take the reading as soon as possible to avoid other
contaminants. This neutral red test strip will be effective at indicating an abnormally
basic urine pH in the field.

References:
1. Urinary Tract Infections in Adults; No. 12-2097; NIH: November 2011; pp 1-7.
http://permanent.access.gpo.gov/gpo49844/Urinary_Tract_Infections_Adults_508.pdf
(accessed April 15, 2015)
2.Adair, J.; Mehta, Khanjan; Ukweli Test Strips: Improving Livelihoods and Access to
Healthcare through Inkjet Printing Diagnostics; HESE PSU: pp2.
3. Mundt, L.; Shanahan, K.; Chemical Analysis of Urine. Graffs Textbook of Routine
Urinalysis and Body Fluids [Online]; Goucher, J., Eds.; Lippincott Williams & Wilkins:
Philadelphia, 2011; pp 36-39. https://cms.psu.edu/section/default.asp?
id=201415SPUP___RCHEM_112H001 (accessed April 15, 2015).
4. Drekonja, D. M.; Johnson, J. R.; Urinary Tract Infections. Management of
Antimicrobials in Infectious Diseases: Impacts of Antibiotic Resistance [Online];
Mainous, A. G., Pomeroy, C., Eds.; Humana Press: New York, 2010; pp 183-184.
http://link.springer.com/chapter/10.1007%2F978-1-60327-239-1_10 (accessed April 15,
2015).
5. Neutral Red. Oxford Dictionary of Biochemistry and Molecular Biology [Online];
Atwood, T., Cammack, R., Campbell, P., Parish, H., Smith, Stirling, J., A., Vella, F., Eds.;
Oxford University Press: 2008.
http://www.oxfordreference.com.ezaccess.libraries.psu.edu/view/10.1093/acref/97801985
29170.001.0001/acref-9780198529170-e-13566 (accessed April 15, 2015).
6. Chen, S. M.; Lin, K. C.; Lin, Y. C.; Electrocatalytic reaction of hydrogen peroxide and
NADH based on poly (neutral red) and FAD hybrid film. Analyst [Online] 2012, 1, pp
137, 186-194. http://pubs.rsc.org/en/content/articlehtml/2012/an/c1an15739f (accessed
April 15, 2015).
7.Acree, S. F.; Bates R. G.; pH Values of Certain Phosphate-Chloride Mixtures, and the
Second Dissociation Constant of Phosphoric Acid From 0 to 60C . J. National Bureau
of Standards [Online] 1943, Volume 30, pp 130-131.
http://nvlpubs.nist.gov/nistpubs/jres/30/jresv30n2p129_A1b.pdf (accessed April 15,
2015).
8. Burdge, J.; Overby, J.; Acids and Bases. Chemistry: Atoms First; The McGraw-Hill
Companies, Inc.: Boston, 2012; pp 656-699.

9. Putnam, D. F.; Composition and Concentrative Properties of Human Urine; NASA

Contractor Report - 1802; NASA: Washington, D.C., July 1971; pp 38-39.
http://ntrs.nasa.gov/archive/nasa/casi.ntrs.nasa.gov/19710023044.pdf (accessed April 15,
2015)
10. Bronze, M. S.; Gonzalez, G.; Proteus Infections Workup. Medscape [Online]; Glatt,
A,, Talavera, F., Eds.; 2014. http://emedicine.medscape.com/article/226434-workup
(accessed April 15, 2015).
11. MedlinePlus. http://www.nlm.nih.gov/medlineplus/ency/article/003616.htm,
http://www.nlm.nih.gov/medlineplus/ency/article/003610.htm (accessed April 16, 2015).