the fiber surface [ 43 ]. Since cellulose has a slightly anionic surface, it can serve as a
scaffold for immobilizing positively charged bio-receptors (enzymes, antibodies,
and nucleic acids). Covalent bonding of bio-receptors is often exploited for its
robust attachment of biomolecules through EDC/NHS chemistry.
There are many techniques reported in the literature for fabricating paper-based
microfluidic devices including: (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
treating. The fundamental applications of all these techniques is to create hydro-
phobic barriers onto the sheet of hydrophobic cellulose matrix, which will consti-
tute the micron-sized capillary channels. To prevent leakage of the applied solution
and keep it in the fluidic channels, the paper materials are often coated with a
polypropylene layer (or other plastics). A variety of hydrophobic materials such as
photoresist SU-8, wax, or alkyl ketene dimer (AKD) are widely used to create
fluidic channels inside the paper [ 42 ]. Depending on the hydrophobic agents
employed, the paper pores can be either blocked (after using SU-8) or covered by
layer of physical deposition (polystyrene or wax); in some cases the cellulose fibers
can be chemically modified (after using AKD).
In general, four detection methods have been reported for the detection of
analytes in paper-based microfluidics: (1) colorimetric detection, (2) electrochemi-
cal detection, (3) chemiluminescence detection, and (4) electrochemiluminescence
detection. Colorimetric detection protocols are typically related to enzymatic or
chemical color-change reactions. In most cases, the analysis of results can be
visually assessed, which is adequate when a yes/no answer or a semi-quantitative
detection method is sufficient for diagnosis [ 41 ]. Electrochemical detection has
higher sensitivity, enabling the detection and quantification of analytes even in the
nano-Molar (nM) range. Most studies to date have been focusing on exploiting the
colorimetric detection and the electrochemical detection of biomarkers in paper-
based microfluidics. Chemiluminescence and electrochemiluminescence are the
most common optical detection methods in microfluidics. They are performed in
the dark and therefore exposure to ambient light will yield inaccurate readings.
However, they have not been widely used in paper-based microfluidics; only a few
studies investigated using these two detection methods for detecting analytes in
paper-based microfluidic devices or paper-based microarray plates [ 43 , 44 ].
Although paper-based microfluidics are predominantly used in recent days, they
still have issues. They are:
- Low efficiency of sample delivery to the sensing surface due to the retention of
samples within the paper fluidic channels and sample evaporation during trans-
port. In most of the cases, the volume that reaches the detection zones verses the
total volume within the device is usually less than 50 %. This makes the
application of paper microfluidics difficult to utilize in clinical diagnosis situa-
tions where the sample quantity is tiny or the sample is costly. - The micro-channels (barriers) patterned with hydrophobic materials are not
hydrophobic, or strong, enough to withstand samples of low surface tension.
158 P. Manickam et al.