Low-Cost Printed Test Strips for Diabetes in Developing Countries

Low-Cost Blood Glucose Monitoring System with Printed On-Demand Test Strips for
Implementation in Resource-Poor Settings
Joseph Wilson
South Carolina Governor’s School for Science and Mathematics

ABSTRACT
More than 285 million people worldwide are diabetic and require daily blood glucose
monitoring. As glucometers have evolved, they have become more accurate (~3% variance), but
test strips can be expensive for patients, especially those without health insurance. In addition, in
developing countries that rely on donated medical supplies, matching meters and strips are not
always available to patients. The goal of our project is to design a low-cost meter and strip
system that can be used in resource poor settings when standard meters or strips are not
available. Our strategy is to create test strips that may be printed on-demand by a standard inkjet
printer. To print the enzyme, we used emptied color-ink cartridges. Glucose oxidase, horseradish
peroxidase, and o-dianisidine dihydrochloride are inserted in the printing wells of the cartridge.
These enzymes catalyze a glucose reaction whose final products elicit a color change. The
enzymes are printed using a template in Microsoft Word. By varying the color in the templates,
we can select the amount of each enzyme applied to the paper. To read the strips, we designed a
low-cost glucometer using LED lights, a photodetector, and an amplifier that outputs the
absorbance, which is processed by an Arduino microcontroller to determine the glucose
concentration based on a standard curve. For proof of concept, the strips were tested using
glucose solutions of varying concentrations (0-450 mg/dl). The absorption measurement was
able to distinguish between glucose solutions with 25 mg/dl accuracy.
INTRODUCTION
Diabetes is not only a problem in first-world countries, but it is also becoming a silent
killer in resource-poor third-world countries. The number of cases in developing countries is
expected to more than double in the next 20 years, expected to explode from 115 million cases in
2000 to 284 million in 2030 (World Health Organization). Of the current 285 million cases of
diabetes worldwide, over 70% of cases occur in low to middle income countries. People must
first be diagnosed with diabetes; however, only half of the patients in the third-world are given a
diagnosis. Even if patients are diagnosed, they must monitor their blood glucose many times
daily and take the appropriate action to either raise or lower their sugar, which becomes very
challenging in resource-poor settings, where glucometers are in short supply. If patients cannot
monitor their blood glucose levels, the rate of complications and mortality rises exponentially
(“Diabetes Facts”).
Developing countries heavily rely on donated supplies, which can be problematic for
those who suffer from diabetes. Supplies for glucose monitoring are not always available, and

when they are, the quantity is limited, which does not guarantee treatment and monitoring for all
individuals. Even if hospitals and clinics receive testing supplies for those who are diabetic, these
supplies may not be usable. Most glucometers sold in developing countries require a specific test
strip. Many times, clinics will receive one brand of glucometer and another brand of test strip,
rendering both technologies virtually useless for the patient. In addition, due to issues of enzyme
stability, the strips have a limited shelf life and may expire before they are donated. The lack of
these resources results in many medical complications that could be easily prevented with access
to testing supplies.
To solve this problem, a cost-effective means of monitoring blood glucose must be
designed to be implemented in resource-poor settings to simplify the treatment and diagnosis of
diabetes. A device such as this would provide an accurate indication of blood glucose levels in
places that could not obtain these data before. This device would allow for doctors to prescribe
medications and treatments for those with diabetes and help prevent complications and
hospitalizations that take up needed bed space in clinics and hospitals.
A cost-effective means of monitoring blood glucose would reduce premature deaths from
unmonitored cases of diabetes. This device would provide doctors and patients with an effective
and obtainable means of monitoring blood sugar on a regular basis to manage diabetes. One must
take many steps in one’s treatment of diabetes, but to start taking these steps, one must be able to
monitor one’s blood glucose levels. The development of a system such as this is the biggest step
in helping patients in the developing world manage living with diabetes.
The goals in this project are (1) to develop a method to print test strips on-demand on
regular paper using a regular inkjet printer with modified cartridges filled with enzyme and (2) to
produce a user-friendly colorimetric meter for the strips that may be easily assembled in the
third-world that is accurate in the range of 0 to 450 mg/dl of glucose. These two elements would
combine to create an inexpensive, easily-obtainable method of managing and treating diabetes in

resource-poor settings. The glucometers could be assembled near the clinic and test strips could
be printed as needed in the clinic lab. The device would provide an accurate qualitative measure
of blood glucose and would be able to show the user if he or she were out of the normal range
and needed to take action. Ideally, this device would provide different kinds of feedback,
whether the patient is in a normal or abnormal range.
The design idea is to use a regular thermal inkjet printer to “print” enzymes onto paper on
an as-needed basis. By using a printer and template to print the strips, a uniform amount of
enzyme will be applied. Though the device must be cost-effective, it must also be reliable. The
use of a printer provides a means of maintaining quality and consistency among the strips and is
simple to use. In this study, the Epson Workforce 30 and H.P. Deskjet 500 printers were used,
which would be readily available in developing countries because of the low cost of this inkjet
technology. The Epson is a newer model whose cartridges are harder to modify than the H.P.’s
cartridges. The Epson has separate color cartridges while the H.P. has one color cartridge with
different compartments for magenta, cyan, and yellow respectively. The Epson has a chip to
monitor ink levels and render cartridges unable to be reused after being emptied, which initially
caused problems; however, the older H.P. cartridges do not contain this chip technology.
Most meters commercially sold today detect glucose levels through an electrochemical
mechanism where the glucose concentration is converted into a voltage or current signal using
special sensor strips (Wang). Recently, microfluidic paper-based analytical devices (mPADS)
have been developed specifically for use in developing countries. These systems tend to be
colorimetric. Paper-based systems are important because paper is widely available, affordable,
compatible, and easily shows a color change because of its white color. The mPADS have
complex systems of hydrophilic microchannels surrounded by hydrophobic barriers to control
the amount of blood allowed to react with the enzyme. The length, width, and height of the
channels are determined by the type of paper used. The reagents used for running the assay of

glucose to determine concentration are then printed onto the paper with an inkjet printer.
Quantitative colorimetric detection of different analytes using mPADS has been achieved
through the process of reflectance detection, where the amount of light reflected off the surface
of the test strip is a function of the concentration of the analyte (Whitesides). A camera captures
the reflected light and the intensity of the color is used to calculate concentration based on a
calibration curve. This method uses the same color change that traditional colorimetric
biosensors utilize. Whitesides et al. successfully quantified glucose concentration in urine using
these methods, which is shown below in Figure 1 (Whitesides).
Figure 1. mPADS for analysis of glucose in urine from Whitesides et al. A) Patterning of paper
shown using Waterman red ink to illustrate integrity of hydrophilic channel. B) A complete
mPAD after depositing the reagents. The left bulb was prepared for glucose detection, the right
bulb was being used for protein detection assays. C) Positive assays for glucose seen by the red
color on the left of the mPAD. D) The left portion depicts results of paper based glucose assays
using a range of concentrations in artificial urine. E) Analytical calibration plot for glucose
concentration.
A colorimetric biosensor was utilized. A biosensor is an analytical device that uses
specific biochemical reactions to detect compounds in a biological sample. This is usually
accomplished by converting a biological response into an electrical signal (Chaplin). A glucose
biosensor that operates based upon oxidation-reduction reactions between glucose, glucose
oxidase (GOx), horseradish peroxidase (HPOD), and O-dianisidine dye was used. The reaction is
shown here:

𝒈𝒍𝒖𝒄𝒐𝒔𝒆 + 𝑯 𝟐 𝑶 + 𝑶 𝟐 𝒈𝒍𝒖𝒄𝒐𝒏𝒊𝒄 𝒂𝒄𝒊𝒅 + 𝑯 𝟐 𝑶 𝟐
𝑯 𝟐 𝟎 𝟐 + 𝒐 − 𝒅𝒊𝒂𝒏𝒊𝒔𝒊𝒅𝒊𝒏𝒆 𝟎 − 𝒅𝒊𝒂𝒏𝒊𝒔𝒊𝒅𝒊𝒏𝒆 + 𝑯 𝟐 𝑶
(reduced form) (brown color)
The final reaction between o-dianisidine, hydrogen peroxide, and horseradish peroxidase
produces a color change (detectable at a range of about 400-600nm) based upon how much
glucose is present in solution. This subtle difference in color and absorbance between different
glucose concentrations is the basis of the colorimetric biosensor in this research.
This enzymatic technique has become commonplace in glucose monitoring systems. A
photodetector was used to output a difference in voltage, which may then be converted into a
measure of absorbance. Using a standard curve obtained from running assays of glucose from
concentrations of 0 mg/dl to 450 mg/dl, an absorbance may be converted into a concentration. A
color change in blood occurs because of the reaction between blood glucose, enzymes, and a dye.
This change in color and also absorbance will be detected by a photodiode in the glucose meter.
The meter must meet specific performance specifications. The glucometer must be able
to accurately show blood glucose levels that someone who is diabetic may experience. This
ranges from lows close to 0 mg/dL to as high as 450 mg/dL. The test strips also must be able to
have a decent shelf life. At minimum, they must be viable for 24 hours after the enzyme
solutions are deposited.
METHODS AND MATERIALS
Dilutions of glucose were made from 45% glucose solution (450 g/L) (MediaTech Inc.)
in distilled water. A stock of 450 mg/dL was prepared using the 45% glucose solution and
Peroxidase
Glucose Oxidase
30 Minutes
Figure 2: The
color changing
reactions at
time 0 and 30
minutes.

distilled water. This stock was left to sit and mix for two hours. From the stock of 450 mg/dL,
concentrations from 0-450 mg/dL (by 25 mg/dL) were created for the purpose of creating a
standard curve.
Using a procedure based upon Sigma’s Enzymatic Assay of Glucose, solutions were made
for testing. Sodium acetate buffer (50mM) was prepared by adding 3.402g of sodium acetate
trihydrate (Sigma) to 500mL of deionized water. The pH was then adjusted to 5.1 using
hydrochloric acid (Acros Organics).
O-dianisidine solution (0.21 mM) was prepared by dissolving 20mg of o-dianisidine
dihydrochloride (Sigma) in 8mL of purified water in a vial protected from light.
GOx solution (0.8 unit/mL) was produced by adding 6.94 mg of Type II glucose oxidase
from Aspergillus Niger (17,300 units/g ) (Sigma) to 150mL of cold 50mM sodium acetate buffer.
POD (60 units/mL) was prepared by adding 46.6mg of Type II horseradish peroxidase
(193 purpurogallin units/mg sold) (Sigma) to 150mL of cold water.
A standard 96-well plate was obtained to run assays of differing concentrations of
glucose to obtain a calibration curve. Assays were run using different amounts of glucose, dye,
enzyme, and buffer, which is shown in Figure 3; furthermore, each combination was run in
duplicate or triplicate.
Assay # Amount of
Enzyme
Amount of
Buffer
Amount of
Dye
Amount of
Glucose
1-5 200µL * * 10µL
6-9 11µL 282µL 7µL 10µL
10 50µL 203µL 7µL 10µL
11 75µL 150µL 10µL 10µL
12 100µL 100µL 10µL 10µL
* These assays were run with the Sigma Enzymatic Assay of Glucose kit, which combined
dye, buffer, and enzyme.
Figure 3: The
relative amounts
of enzyme in
each well for all
of the assays.

The buffer was first deposited in well A1 and advancing until A12 was reached. Rows B, C, E,
F, and G were filled in the same way. Row D was skipped to ensure that overflow would not
contaminate the different samples in vertically adjacent wells. This filling procedure was
repeated with GOx, HPOD, dye, and different concentrations of glucose. Each concentration of
glucose would be in three wells, all vertically adjacent to one another in order to run triplicate
samples on each plate. Concentrations of glucose were increasing in increments of 25 mg/dL
starting at 0 mg/dL in wells A1, B1, and C1. When adding glucose, pipette tips were switched
between each concentration of glucose to avoid contamination.
Once everything was added to the 96-well plate, the enzymes were allowed to react for
30 minutes at room temperature before being put into the spectrophotometer. The
spectrophotometer was set to read at 500 nm and also a spectrum of 400-600 nm by 20 nm. At 45
minutes, the plate was again read at 500 nm. All of this data was imputed into Microsoft Office
Excel 2010™ from the Gen5™ spectrophotometry program. This data was graphed using an
average of each concentration and formulating a line of best fit. The R2 value was also examined
for the accuracy of the curve. The curve from the ninth assay is shown in Figure 4.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 100 200 300 400 500
Absorbtion
(Concentration [in mg/dl])
Glucose Assayat 500nm
Figure 4: The
standard curve
obtained from the
data of the 9th
Assay of glucose.
The Arduino
microprocessor
was programmed
using the equation
y=.0017x + .0121
to convert light
absorbance into
glucose
concentration,
with an R² value
of 0.9579.

Before printing test strips, different designs, combinations of enzymes, and types of paper
were tested. The main problem encountered was the viscosity of blood and how blood could be
applied to the strip in a sterile manner. To help influence the flow of blood to the correct area on
test strips, various hydrophobic materials were used and tested. To test patient-to-strip delivery
systems, a hydrophobic sheet was obtained, such as contact paper or parafilm. An absorbant
paper, such as filter paper, was backed with the hydrophobic sheet. Then designs were cut into
the filter paper without cutting away the hydrophobic surface using an X-ACTO knife. A
mixture of corn starch and water with a viscosity similar to that of blood was applied using a
gloved hand. With a sweeping motion, a finger prick was simulated and the distance the corn
starch and water mixture moved over a thirty minute period was observed. After obtaining a
design that worked, strips were printed using the printer.
To make test strips with the printer, modified cartridges with the ink removed were used
to print enzyme onto regular paper. An old H.P. Deskjet 500, which had ink cartridges that were
easy to clean and fill, was first used. A newer Epson Stylus Workforce 30 was then used because
it is currently widely available in the market and is relatively cheap. The Epson has cartridges
that are very hard to clean, fill, and that reset themselves after they become empty. Therefore,
instead of cleaning and emptying the Epson cartridges, Epson-compatible cartridges that were
reusable, refillable, and came empty were purchased for ease of use. However, for both printers’
cartridges the same care techniques were used.
Before inserting enzyme and using the cartridges, they were cleaned to prevent clogging
and get rid of extra salts and proteins. The cartridges were immersed in a 1:1 deionized water
solution of rust inhibitor for 10 minutes (Burnishine Products). The cartridges were immersed in
a 1:4 in deionized water instrument lubricant solution for 30 minutes (Burnishine Products). The
cartridge was put in a beaker full of deionized and sonicated for 15 minutes. After printing, the

cartridges were rinsed out thoroughly. They were then put it in the same lubricant solution as
before for 30 minutes and sonicated for 15 minutes.
To print test strips, the same procedure was followed; however, the templates for printing
changed over time. To print, the printer was first powered on and given time to warm up. The
desired printing surface was placed in the printer’s tray (wax paper, regular paper, or filter
paper). On Microsoft Word, a template was either created or selected from premade templates
for printing test strips. An example of the test strip design is shown in Figure 5:
The cartridges were filled with 10 mL of glucose oxidase, peroxidase, and o-dianisidine
dye and inserted into the printer. Glucose oxidase was in the magenta cartridge, peroxidase was
in cyan, and o-dianisidine was in yellow. In Microsoft Word, the file was selected to print on the
printer. The printer was warmed up again and the cartridges moved to the “ready” position. In
the old printer, the paper feed mechanism had to be bypassed by pulling on it. This tricked the
printer into thinking regular sized paper was inside it, so that the printer would actually print on
paper or wax of any size. For multiple copies, the paper feed mechanism would have to be
manually bypassed each time.
Once test strips were printed, glucose could then be applied to the strips and the
glucometer could be used to find the absorbance of the strip. The absorbance directly relates to
the level of glucose. To test the absorbance of the strips, 5µL of glucose solution is first applied.
After waiting a few minutes and observing a color change on the strip, the strip is put on the
photodiode of the glucometer. On the other photodiode is a strip with glucose solution and all
Figure 5: The design of our
test strips. The enzyme is
printed in blocks on the ends
of the strip. The wax paper
causes blood to move and
interact because of capillary
action.

enzymes except for glucose oxidase. When the glucometer runs, an LED light shines onto the
strip and photodiode. The photodiode compares the absorbance to the other diode, which acts as
a control, and outputs the absorbance to a LOG102 amplification chip. The amplification chip
then outputs to an Arduino microprocessor which uses the equation determined from the
standard curve and converts the absorbance to a concentration of glucose.
RESULTS
A calibration curve with an R2 value of .9571 was obtained through the running of assays
and their analysis in Microsoft Excel 2010. An R2 value of over .9 is said to be statistically
significant. Because this value is over .9, the glucometer should be relatively accurate in a range
of 0-425mg/dl of glucose and is precise to 10mg/dl intervals.
Test strips have been created by layering filter paper and contact paper. Test strips were
designed so that blood would wick down the test strip in a controlled manner. This way, the
amount of blood on the strip initially would not matter unless it was an amount too small to be
detected. Test strips have been successfully printed using the printing process described
previously. The test strip design is currently a 5mm wide and 20mm long filter paper and
contact paper strip with printed o-dianisidine, glucose oxidase, and horseradish peroxidase.
When fetal bovine serum is applied to the test strips, the color changing reactions occur
in about thirty minutes. The glucometer will read the difference in voltage and then convert this
into a concentration of glucose that is, on average, only 6mg/dL off. However, the readings have
a standard deviation of over 40.0mg/dL.
DISCUSSION
The color-changing reactions have been taking 30 minutes to complete, which is too
much time because the patient has to take action quickly. Modern meters take seconds to give the
user a reading.

In addition, modern meters are accurate up to concentrations of 600mg/dl and are very
precise.
This glucometer and test strip method is relatively inexpensive, utilizing cheap materials
such as filter paper, contact paper, and parts widely available.
Even though this meter does not currently comply with ISO standards, it may be
employed in developing countries when other meters are not available. The creation of a system
such as this is integral for resource-poor nations.
This device is to be used as a bridge for those patients who are waiting for more accurate
testing supplies.
Although this meter-strip system is currently not as accurate as standard commercial
systems and further tests are required to improve the design, it is still accurate enough to inform
patients of their relative range of blood glucose (low, normal, high). This can allow them to still
take appropriate action for raising or lowering their blood glucose. Future work will focus on
optimizing the strip design to shorten the time necessary before a measurement is made and to
decrease the variability between measurements. In addition, further testing will be performed to
assess the stability of printed strips. The hope is that this system can be implemented in resource
poor settings where glucometers are in short supply and help decrease the incidence of diabetes
related complications in these settings.
Acknowledgments
I would like to acknowledge Dr. Delphine Dean of the Biomedical Engineering
Department at Clemson University for serving as my mentor and all of her help throughout the
research process. I would also like to acknowledge Kayla Gainey and Kelsey Byrd as my
partners on this project.

Literature Cited
“Diabetes Facts”. World Diabetes Foundation Online. Web. 26 January, 2012. <
http://www.worlddiabetesfoundation.org/composite-35.htm>
“Diabetes cases could double in developing countries in the next 30 years”. World Health
Organization Online. Web. 26 January, 2012. http://www.who.int/mediacentre/news/
releases/2003/pr86/en/
American Diabetes Association. Diabetes Care. Web. 11 July, 2012.
Chaplin, Martin. “What are biosensors?”. London Southbank University Department of
Engineering and Science. Web. 23, April 2012. <http://www.lsbu.ac.uk/biology/enztech/
biosensors.html>
Wang, Joseph. “Electrochemical biosensors”. American Chemical Society: Chemical Reviews,
2008, 108(2): 814-825.
Whitesides, George. “Diagnostics for the Developing World: Microfluidic Paper-Based
Analytical Devices. Analytical Chemistry, 2010, 82(1): 3-10.

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