a microfluidics experiment for the quantitative analysis

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