CL 625 Term Paper

Paper Based Microfluidics

Group 16
Shreya Mukherjee, Sandip Kumar Sarma, Prince Baranwal
Gaurab Sarkar, Shirsendu Mitra, Dumbi Boipai

Abstract
Paper based microfluidics has been of enormous essence in point of care diagnostics. This report focuses on the utility of paper-based
devices in various sectors and how they can be quantified. A mathematical model of the capillary flow through the paper is also shown. We
will see how energy balance will provide us with a non-linear partial differential equation to model the system. We will also see how the results
obtained can be quantified through various methods like colorimetry. The fabrication processes will be discussed in detail. The future prospects
of paper-based devices are also pondered upon.

1. Introduction

2. Fabrication of PADs

Paper as a substrate for micro-fluidic assays was largely

ignored until 2007 when Martinez et al. [1] reported the first microfluidic paper based analytic device for chemical analysis. Besides
being cheap paper has its own advantage of power free fluid
transport via capillary action, a high surface to volume ratio and the
ability to store reagents in active form within its fibre network.
Paper has several additional advantages as a material for
making point-of-care diagnostic devices: (i) paper is thin,
lightweight, available in a wide range of thickness, and easy to stack,
store, and transport; (ii) paper is made of cellulose or cellulosepolymer blends and is compatible with biological samples; (iii)
paper can be modified chemically to incorporate a wide variety of
functional groups that binds with bio-molecules, (iv) paper is usually
white and has strong contrast with coloured analytes, it performs
well for colorimetric tests; (v) paper is flammable, so used PADs
can be disposed off easily and safely by burning them; (vi)
Functionalities can be by adding additives during manufacturing.
However, paper also has several disadvantages as a
material for diagnostic devices. Since paper fibres scatter large
amount of light, it performs poorly in absorbance tests. The wicking
rate of paper is not uniform, and the distance a fluid will travel along
a channel in paper is proportional to the square root of time. Hence,
longer channels may not be practical.

The earliest paper-based tests used paper, manually cut

into sections or strips and treated with a chemical reagent that
reacted with an analyte to produce a colored product. Although easy
to fabricate, realization of more complicated devices with enhanced
functionality has been pursued. The following techniques can be
used for fabricating paper based micro-fluidic devices: (1)
photolithography, (2) plotting with an analogue plotter, (3) ink jet
etching, (4) plasma treatment, (5) paper cutting, (6) wax printing, (7)
ink jet printing, (8) flexography printing, (9) screen printing, and
(10) laser treatment, etc. The basic principle behind these fabrication
techniques is to pattern hydrophilic-hydrophobic contrast on a sheet
of paper in order to create micro-nano scale (i.e., hundreds to
thousands of ms) capillary channels on paper.

1.1. Major Breakthroughs

Martinez et al. [1] first demonstrated paper-based
microfluidic devices for the colorimetric detection of glucose and
protein. The investigation was based on a change in colour when
introduction of the sample filled the reaction zone. Ratnarathorn et
al. [2] demonstrated the colorimetric detection of copper using silver
nanoparticles (AgNP) on paper devices. Arena et al. [3] investigated
the response of a paper-based sensor for ethanol detection. The
sensor was fabricated by integrating multi-walled carbon nanotube
electrodes with an indium tin oxide nanoparticulate powder-sensing
layer on paper. The addition of a binder promoted the adhesion of
the indium tin oxide nanoparticulate powder to the glossy paper
substrate. Subsequent analysis of ethanol was carried out in air by
measuring the current upon applying triangular applied voltage.
Steffens et al. [4] developed a low cost gas sensor using graphite
interdigitated electrodes coated with a thin sensing layer of dopedpolyaniline. Changes in conductivity were observed in response to
the presence of nitrogen gas.

Figure 1: (A) Schematic of a paper based micro-fluidic channel; (B)

and (C) Patterning by Photolithography; (D) and (E) Patterning by
Wax Printing.
Photolithography on paper requires six steps and produces
well-defined hydrophobic barriers of photoresist that extend through
the thickness of the paper. An example of a device fabricated by
photolithography with a central channel that wicks fluids into three
independent test zones is shown in the figure 1 (B) and 1 (C). Wax
printing, on the other hand, requires two steps and produces
hydrophobic barriers of wax that extend through the thickness of the
paper. When the paper is heated, the wax melts and spreads both
vertically and laterally into the paper. The vertical spreading creates
the hydrophobic barrier. The lateral spreading lowers the resolution
of the method and produces barriers that are much larger than the
original printed pattern [5]. An example of a device fabricated by
wax printing with a central channel that wicks fluids into three
independent test zones is shown in the figure 1 (D) and 1 (E).

3. Mathematical Modelling

H 0 0

(16)

In order to find the governing equations for capillary rise

through paper a force balance was made by Masoodi et al [6]. The
6kinetic, gravity, viscous and surface tension forces were balanced.
The various forces as described can be formulated as given below.
The gravitational force is the weight of the liquid column
inside the capillary tube and can be expressed as:

dH 0
0
dT

(17)

Fg gAh t

(1)

The kinetic force acts on the column of liquid, which

depends on mass and velocity of the liquid. Since both mass and
velocity of liquid column change with time, so:
h t

mu A h t

t
t
t

Fk

3.1. Results and Discussion

The numerical solution and the experimentally obtained
results were compared by Zhmud et al. for = 1.4 and by Quere et
al. for = 21.4 [6]. The accuracy of numerical results is very good
at the beginning but the error increases slightly as time passes. It
should be related to the neglected terms/effects, such as the entrance
effect, the difference between descending and ascending contact
angles, and Vena contracta effect.

(2)

The viscous and surface tension forces can be described as follows:

8 Ah t h t
R2
t

FV

(3)
(4)

Fs 2 r C

The viscous force term is developed from Hagen-Poiseuilli equation

given by:

8 LQ
R4

(5)

Now the force balance and the subsequent substitution give the
following set of equations:
(6)
F F F F
s

C gAh t A

h t 8 Ah t h t

h t

t
t
R2
t

h t 8 h t h t

Pc gh t h t

t
t
R2
t

(7)

(8)

Where Pc is defined as the capillary pressure

Non-dimensionalization of equation (8) gives the
following set of equations and then equating the co-efficient to unity
we the values of equilibrium height (he) and mean residence time ()
in term of known parameters.
h
he

T
1

(9)

he 2 dH ghe
h 2 d dH
H

H e2
H

2
Pc R
dT
Pc
Pc dT dT

4. Detection of analytes using PADS

4.1. Colorimetric Analysis
Colorimetric detection of analytes is one of the most used
detection technique. The immunoassays consist of a strip of paper
with sample pad for introduction of sample, reagent pad which
contains antibodies conjugated to a single indictor which are specific
to a target antigen. As the sample is introduced to the sample pad, it
migrates along the paper strip via capillary forces where the presence
of antigen in the sample binds to signal antibody.

Pc
g

(11)
(12)

8 Pc

gR

2 gR 4
64 2 he

(13)
(14)

The final non-dimensional form of equation looks like below:

H

Figure 2 shows the numerical prediction for small values

of . Since the differences between different curves are significant
for short times, so the results are restricted to T < 2. When = 0, the
predictions of Eq. (15) matches predictions obtained by Lucas
Washburn Equation (LWE).

(10)

he

Figure 2: Oscillatory behaviour of liquid column.

dH
d dH
H
H
1
dT
dT dT

(15)

The above non-linear partial differential equation can be

solved using 4th order Runga-Kutta method. The initial conditions
for solving the equation are:

Figure 3: (A) Colorimetric analysis and (B) Electrochemical

The intensity of the colour that develops in the test zone is
a function of the concentration of the analytes (Figure 3 (A) [1]). The
reflected light can be captured by a digital camera or smart phones
[7]. Although smart phones are superior to flatbed scanners in
regards to portability, they suffer from changing ambient light
conditions, rendering image intensities inconsistent. Recently,
several groups addressed this problem by developing intensity
correction software for smart phones or by creating devices to
physically block ambient light during image acquisition.

4.2. Electrochemical Detection

Electrochemical techniques often require a three-electrode
system, that is, a counter electrode, reference electrode and working
electrode. In creating a paper-based electrochemical sensor, a threeelectrode system is replicated on paper (Figure 3 (B)). Lankelma et
al. [8] created a new design for a paper-based electrochemical
system for flow injection analysis.
4.3. Chemiluminescent Detection
Yu et al. developed a microfluidic paper-based device for
detection of glucose and uric acid based on the chemiluminescence
reaction between a rhodamine compound and hydrogen peroxide.
The analytes are introduced through an injection hole and travels
through two bioactive channels, where the enzymes (glucose oxidase
and urate oxidase) are deposited, and reach the two
chemiluminescent detection areas containing the rhodamine
derivatives [7].

5. Applications
5.1. Biomedical: Enzymatic Methods
Glucose is one of the most important indicators for
diagnosis of diabetes. Enzymatic methods have been widely used for
colorimetric detection of glucose. Zhu et al. [9] demonstrated
detection of glucose using a tree-shaped PAD with 2,4,6-tribromo3-hydroxy benzoic acid and 4-aminoantipyrine. They also showed
usage of three-dimensional PADs for simultaneous detection of
glucose and protein in urine. Pollock et al. fabricated a 3D PAD for
semi quantitative measurement of alanine aminotransferase and
aspartate aminotransferase which can be used for liver function tests
[10].
5.2. Biomedical: Immunoassays
PAD based immunoassays offer a simple and cost
effective solution for point-of-care monitoring. Apilux et al.
demonstrated that by creating flow paths of different lengths,
complex multistep ELISA tests can be automated in PADs. He
studied effectiveness of various delaying patterns printed on
nitrocellulose membrane and showed higher effectiveness for hCG
detention than conventional assays [10].
5.3. Biomedical: Electrochemical
While use of PADs with electrodes printed with
conductive has been demonstrated for glucose detection, they have
a much wider potential. Ge et al. showed that D-glutamic acid, a
neurotransmitter associated with brain damage can be detected with
high sensitivity using GNP coated cellulose fibres with electropolymerized molecular imprint polymer on the surface [10].
Decrease in hexacyanoferrate oxidation due to adsorption of Dglutamic acid on the electrode surface can be measured using
differential pulse voltammetry and showed promise of subnanomolar detection. Researchers have also demonstrated detection
of low-abundant cancer biomarkers using electrochemical signal
enhancing tags.
5.4. Environmental
Paper-based approaches for environmental monitoring are
attractive because accurate, low-cost monitoring is pivotal for
environmental applications. Colorimetric PAD have been
developed for detection of metal ions for environmental monitoring.
PAD have also been developed for non-metal environmental
analytes.
For
example,
a
paper
sensor
to
test

for the presence of nitrite and nitrate in drinking water was

presented by Jayawardane et al. Electrochemical methods
have also been developed for coupling the advantages
of paper substrates with electrochemistry. For example, PADs with
graphite electrode for detection of p-nitrophenol have been
developed [10].
5.5. Food and Beverage Industry
PADs offer a low cost and simple on-site solution for
detection of food contaminants. Colorimetric tests using enzymatic
methods for detection of bacteria in food has been developed [10].
Electrochemical detection of heavy metals and pesticides in food and
beverages has also been developed.

6. Conclusion and Future Scope

The low cost, simplicity, flexibility, and the environment
friendly nature of PADs makes it a promising starting point for the
development of new solutions to the problem of health-relevant
assays in the developing economies. This concept can also be used
in environmental monitoring and in detection of food and beverage
contamination. We hope that in the near future PADs will provide
a platform with several new capabilities for bioanalysis, such as:
1. The ability to store, mix, and combine reagents.
2. The ability to filter samples and separate mixtures into
individual components for analysis.
3. The ability to automatically analyze controlled volumes of
sample starting from unknown volumes of sample.

7. References
[1] Martinez A.W., Phillips S.T., Whitesides G.M., Carrilho E.,
Diagnostics for the developing world: Microfluidic paper-based
analytical devices. Anal. Chem. 2010, 82, 310.
[2] Ratnarathorn N., Chailapakul O., Henry C.S., Dungchai W.,
Simple silver nanoparticle colorimetric sensing for copper by paperbased devices. Talanta, 2012.
[3] Arena A., Donato N., Saitta G., Bonavita A., Rizzo G., Neri
G., Flexible ethanol sensors on glossy paper substrates operating at
room temperature. Sens. Actuators B: Chem. 2010, 145, 488494.
[4] Steffens C., Manzoli A., Francheschi E., Corazza M., Corazza
F., Oliveira J.V., Herrmann P., Low-cost sensors developed on
paper by line patterning with graphite and polyaniline coating with
supercritical CO2. Synth. Met. 2009, 159, 23292332.
[5] Li Xu, Ballerini D.R., and Shen W., A perspective on paper
based microfluidics. AIP, Biomicrofluidics, March, 2012.
[6] Masoodi R., Languri E., Ostadhossein A., Dynamics of liquid
rise in a vertical capillary tube. J. Colloid Interface Sci. 2013, 389,
268272.
[7] Liana D. D., Raguse D., Gooding J.J., Chow E., Recent
Advances in Paper-Based Sensors. Sensors 2012, 12.
[8] Lankelma J., Nie Z., Carrilho E., Whitesides G.M., Paperbased analytical device for electrochemical flow-injection analysis
of glucose in urine. Anal. Chem. 2012, 84, 41474152.
[9] Zhu W.J., Feng D.Q., Chen M., Chen Z.D., Zhu R., Fang
H.L., Wang W., Bienzyme colorimetric detection of glucose with
self-calibration based on tree-shaped paper strip. Sens. Actuators, B:
Chem. 2014, 190, 414418.
[10] Cate D.M., Adkins J.A., Mettakoonpitak J., Henry C.S.,
Recent Developments in Paper-Based Microfluidic Devices. Anal.
Chem., 2014, 87 (1), 1941.