A Microfluidics Experiment for the Quantitative Analysis Laboratory Erin M. Gross , Michelle E. Clevenger, Kalani Parker, and Connor Neuville
Department of Chemistry, Creighton University, Omaha, NE 68178
Introduction
Background
Experimental Method Results Results
References
During the past decade, the emerging field of microfluidics has
moved to the forefront of science, particularly in analytical
chemistry. It would be beneficial for undergraduate students
utilize microfluidic methods. However, conventional
microfluidic fabrication processes are costly and most
undergraduate institutions do not possess to the required
equipment and facilities. With the recent introduction of paper-
based microfluidic devices for chemical analysis,
undergraduate students can obtain hands-on experience in
microfluidics.
This project converted microfluidic assays recently reported in
the journal Analytical Chemistry1 into an analytical laboratory
experiment. Students were given an unknown “urine sample”
containing both glucose and protein and were asked to
diagnose a patient . This experiment was multidisciplinary and
students were exposed to chemical analysis, bioanalytical
chemistry, medicine and the social issues involved in
healthcare.
The following tables along with an “unknown” artificial urine sample
were given to students for diagnosis.1,2
Student Data. This experiment was performed by seven pairs
of students in Quantitative Analysis Laboratory. The glucose
samples were accurately diagnosed. Undergraduate research
students have been working to improve the protein assay. This
work has included optimization of protein standard solutions,
investigation of solution stability, along with the surfactant and
buffer concentration study. Some example student data,
scanned images, and improved data are shown below.
Protein level Diagnosis
< 30 mg/dL Trace levels, normal
30 mg/dL – 80 mg/dL Possible proteinuria, further testing
>80 mg/dL Possible subnephrotic range
proteinuria, nephrotic syndrome,
further testing
Glucose level Diagnosis
0.17 – 1.4 mM No diabetes
> 1.4 mM Further blood testing
STUDENT LEARNING OBJECTIVES:
Exposure to microfluidics
Proper micropipetting techniques
Standard solution preparation
Critical evaluation of data
Real-world applications of science
Glucose, when reacted with glucose oxidase (GOx) and
horseradish peroxidase (HPOx) in the presence of potassium
iodide, produces a color change from colorless to brown, as
iodide is oxidized to triiodide3.
The protein assay4 utilizes the color change of tetrabromophenol
blue (TBPB) from yellow to blue when TBPB binds to albumin, a
protein that indicates disease if present in urine5.
TBPB TBPB-Albumin
Tetrabromophenol Blue Bovine Serum Albumin
Wax Printing: Fabrication of the microfluidic devices. The device
patterns were designed using AutoCAD LT 2010 drafting software and
were printed using a Xerox Phaser 8560 solid ink printer to produce a
hydrophobic barrier to contain the solutions on Whatman #1
chromatography paper. These devices were subjected to heat in an
oven (150°C, 90 seconds) in order to spread the wax through the
paper. The glucose and protein device shapes were optimized to
ensure the most efficient spread of solutions. The snowflake design
allows analysis of multiple reagents.
Figure 1. Examples of microfluidic devices produced by wax
printing. A) Blank glucose device. B) Protein device with reacted
TBPB. C) Optimization of protein device shape. D) Combined
glucose and protein device for simultaneous analysis.
A B C
Urine Test. An artificial urine solution (pH 6.00) was prepared.6 The
glucose and protein reagents were spotted onto the individual test
zones using a micropipette. Glucose standard solutions were prepared
in the artificial urine solution (Figure 3).14 μL samples were aliquotted
to the middle of the device. The sample was allowed to spread into the
test zones through the channels created by the hydrophobic wax
barrier. Protein standard solutions (Figure 3) were analyzed following
the same procedure as the glucose tests.
Photoshop Analysis2. The analysis feature of Photoshop was used to
quantify the color change produced by the reactions. The devices were
scanned with CanoScan 8800F scanner. The glucose samples were
converted to grayscale and the gray mean intensity was recorded for
each test zone. The protein samples were converted to CMYK color
and were analyzed for cyan mean intensity. Refer to Figure 3 for
calibration curves.
Figure 2. Photoshop
analysis of glucose (left) and
protein (right) test zones.
Students were instructed to prepare, by dilution, standard glucose
solutions of 0.17 mM and 1.4 mM and standard protein solutions of 30
mg/dL and 80 mg/dL . The students prepared the devices, spotting the
protein test zones (circular) with pH 1.8 citrate buffer and TBPB; the
glucose test zones (diamonds) were spotted with a solution of GOx,
HPOx and KI. The standards and unknown solutions were
simultaneously tested. After drying, the test strip was scanned and
Photoshop analysis was completed. The students diagnosed their
unknown by comparing the unknown color change to the color of the
standards. The Photoshop analysis further corroborated each diagnosis
and quantified the unknown solutions.
D-Glucose + O2 GOx Gluconic acid + H2O2
2H+ + H2O2 + 3I- HPOx 2H2O + I3
-
Colorimetric tests were used to estimate the concentration of
glucose and protein in urine samples. The data collected from these
assays were used to create standard curves to quantitate the glucose
and protein in unknown urine samples.
Color Scales
Effect of Surfactants in Protein Priming
Solution
0.0 mM
1.0 mM
5.0 mM
10 mM
Figure 3. Concentration can be estimated using the naked eye
with color scales. Color scales are most promising for determining
relative concentrations during “on-site” testing. Color variance in
the concentrations of glucose (top) and protein (bottom) in urine
can indicate disease states in patients.
y = 10.38x - 2.2751 R² = 0.9858
0
20
40
60
80
100
0.0 2.0 4.0 6.0 8.0 10.0
Avg
. M
ean
In
ten
sit
y
Concentration (mM)
Intensity vs. Glucose Concentration
RSD 0.95%
RSD 5.3%
RSD 4.1%
y = 1.1978x - 0.8743 R² = 0.9837
0
4
8
12
16
20
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Avg
. M
ean
In
ten
sit
y
Concentration (μM)
Intensity vs. Protein Concentration
RSD 2.6%
0.0 μM
3.0 μM
5.0 μM
10 μM
12 μM
Conclusion Paper-based microfluidic devices are a cost-effective means to
provide a microfluidics experiment for undergraduate students. This
experiment exposed students to bioanalysis and microfluidics, while
integrating science and social issues. The experiment was performed
within a single laboratory session. A student survey was administered
to assess if the learning objectives had been satisfied. The survey
results indicate an overall agreement that the experiment was
worthwhile, interesting and challenging.
1. Martinez, A.W.; Phillips, S.T.; Carrilho, E.; Thomas III, S.W.; Sindi, H.; Whitesides, G.M. 2008.
Simple Telemedicine for Developing Regions: Camera Phones and Paper-Based Microfluidic
Devices for Real-Time, Off-Site Diagnosis. Anal. Chem. 80: 3699 – 3707.
2. Davidson, J.K. Clinical Diabetes Mellitus: A Problem-Oriented Approach, 3rd Ed.; Thieme: New
York, 2000.
3. Peele, J.D.; Gadsen, R. H.; Crews, R. 1977. Semi-Automated vs. Visual Reading of Urinalysis
Dipsticks. Clin. Chem. 24: 2242-2246.
4. Pugia, M.J.; Lott, J.A.; Profitt, J.A.; Cast, T.K. 1999. High-Sensitivity Dye Binding Assay for
Albumin in Urine. Journal of Clinical Laboratory Analysis. 13: 180-187.
5. Pugia, M.J., et al. 1998. Comparison of Instrument-Read Dipsticks for Albumin and Creatinine in
Urine with Visual Results and Quantitative Methods. Journal of Clinical Laboratory Analysis. 12:
280-284.
6. Brooks, T., et al. 1997. A simple artificial urine for the growth of urinary pathogens. Lett Appl
Microbiol. 24: 203–206.
The authors would like to acknowledge a
Ferlic Undergraduate Research Scholarship, a
Creighton College of Arts and Sciences
Faculty Development Grant and the Creighton
University Chemistry Department.
RSD 0.53%
y = 8.101x - 0.6684
0
4
8
12
0.0 0.5 1.0 1.5
Two-point Calibration Curve
Concentration (mM)
Intensity Glucose
Conc.
(mM)
Low
Std. 0.71 0.17
High
Std. 10.673 1.4
Unk. 20.573 2.6 Calc’d
2.5 Actual
Me
an
In
ten
sit
y
Figure 5. An example of student data. Left, the curve made from
standard glucose solutions (0.17 and 1.4 mM). Right, results
summary. Bottom, images of test strips.
PROCEDURE DEVELOPMENT & OPTIMIZATION
LABORATORY EXPERIMENT
EXAMPLE STUDENT DATA
D
Tween-20 Conc. (%w/v)
pH 1.8 Citrate
Buffer 0.5% 1.0% 2.0%
0.25 M Χ Χ Χ
0.50 M Χ Χ Χ
0.75 M Χ Χ
1.00 M Χ Χ Χ
Figure 4. In previous protein bioassays, the blank devices were
producing false-positive results. In order to correct this, buffer and
surfactant concentrations were investigated. Other surfactants
were also studied. The solution consisting of 2.0% w/v Tween-20
and 0.75 M citrate buffer most effectively prevented false positive
results.
IMPROVED RESULTS
1.4 mM 0.17 mM
glucose
80 mg/dL 30 mg/dL
protein
0.70 mM gl
100 mg/dL pr
unknowns
2.5 mM gl
20 mg/dL pr
standards standards
2.4 cm
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