1. Microfluidic
immunoassays
as
rapid
saliva-­‐based
clinical
diagnostics
A
Review
on
Immunoassays
Regine
Labog
ABSTRACT
Point-­‐of-­‐care
diagnostics
have
benefited
immensely
from
microfluidic
devices.
Before
the
development
of
microfluidic
immunoassays
for
quantitatively
measuring
disease
through
biomarkers,
common
clinical
diagnostics
were
limited
to
binary
results
for
home
pregnancy
tests,
tuberculosis,
and
influenza.
This
paper
describes
an
advance
in
diagnostics
to
measure
a
biomarker
for
periodontal
disease
in
human
saliva.
This
research
could
be
developed
for
rapid,
reliable
measurement
of
analyzing
disease
markers
in
biological
fluids.

2. Introduction
Peridontal disease affects one or more of the periodontal tissues: alveolar bone,
periodontal ligament, cementum, and gingiva. Unlike other diseases, periodontal disease
is a combination of multiple disease processes that share a common clinical
manifestation. If not treated, it leads to tissue deterioration, loss of connective tissue
attachment, and aleveolar bone loss. Furthering diagnostics research with microdevices
can eventually be used to frequently monitor episodic disease progression, enable early
diagnosis of a disease, or continuously assess therapeutic efficacy.
This paper uses microdevices to find matrix metalloproteinase-8 (MMP-8)1, a
major tissue-destructive enzyme in periodontal disease, in samples of saliva. To improve
the assay’s sensitivity to the enzyme, saliva pretreatment of mixing, incubation, and
enrichment, was included before placing the solution in the quantitative immunoassay.
The microchip electrophoretic immunoassay (µCEI) core of the device is based on
photolithographically fabricated molecular sieving gels to enrich the saliva sample and
later resolve a fluorescent antibody from the MMP-8 antigen-to-antibody complex.
Using microfluidics for point of care applications require a platform that is easy to
use, portable, user-friendly, and cheap. Colorimetric detection can fulfill these
requirments.2
Immunoassays
–
Advantages
Most biological procedures normally require solutions to be in an immobilized,
biochemically active phase.3 Immobilization is key, especially for heterogeneous
immunoassays because it affects specificity and sensitivity. Switching from the
macroscale to microscale depends on three main categories for biomolecular
immobilization: surface modification of microfluidic channel walls, packing microfluidic
channels with biomolecule-bearing beads, and packing microfluidic channels with
biomolecule-bearing porous slabs. For mircofluidic bioanalytical assays that do not use
an immobilized phase, an assay based on the rate of diffusion of antibody-antigen
complexes4 in solution as well as a technique for maintaining beads in place in a
recirculating flowstream without permanently immobilizing them is needed5.

3. Research on portable microfluidic devices for clinical diagnostics is a growing
industry because of its massive potential. These diagnostic devices would have lower
manufacturing costs, decreased sample size (here, a small amount of saliva is more than
enough), reproducible, and greater throughput. With the development of point-of-care
microfluidic diagnostics, clients could perform more complex diagnoses in their own
homes.
Immunoassays
–
Disadvantages
A significant disadvantage for microfluidic immobilization systems is its inherent
irreversibility. A channel surface that has been chemically modified is difficult to
remove, renew, or add an immobilized flexibility. This trait limits the flexibility of device
manufacturing since each device must be made with a specific immobilized biochemistry
for a specific application. These devices also take longer to construct as they are more
complex and the physics for macroscale machines differ from microscale devices due to
the laminar flow present in a microdevice.

4. Peridontal
Disease
Peridontal disease is a
progression of gingivitis and
its main cause is poor oral
hygiene. It destroys the
gingival fibers which are the
gum tissues that separate the
tooth from the peridontal
pocket6. Microorganisms
colonize these pockets and
further inflammate the gum
tissues and bone loss. If it is
not diagnosed and treated in
time, the microbic plaque
calcifies to form tartar and
must be removed above and
below the gum.
The prevalent method for measuring periodontal disease is with a periodontal
probe. It is placed between the gums and the teeth and slipped about 2 to 3mm below the
gum line. A subject with a peridontal pocket deeper than 7mm risks eventual tooth loss
over the years. However, this disease could go on without recognition for many years.
Types
of
Immunoassays
Microarrays are commonly used to perform immunoassays. An immunoassay
typically immobilizes antibodies and exposes them to a biological sample. It is separated
into four different types: direct-binding, sandwich (ELISA), competitive, and
displacement.
Direct-binding is when the antibody is labeled, normally fluorescently, and binds
with the target antigen. This method is not only quicker, but also avoids cross-
contamination with a secondary antibody. However, direct-binding requires using every
antibody which can be expensive and time-consuming. Also, some antibodies may not

5. qualify for direct-binding.
Sandwich (ELISA) quantifies the amount of antigen between the primary and
secondary antibodies. The target antigen must have at least two sites to bind to the
primary and secondary antibody since both must act in the sandwich. This restricts
sandwich assays to antigens with multiple binding sites for antibodies, such as proteins or
polysaccharides. However, sandwich is useful when there are low concentrations of
target antigens or high concentrations of contaminating proteins.
Competitive is used when a target antigen does not have any "matched pair"
antibodies to bind to. Here, the higher the antigen concentration, the weaker the signal
since fewer antibodies will be able to bind to the antigen in the well. The major
advantage is that it can use crude or impure samples to selectively bind any antigen
present. For the purposes of this paper, a competitive immunoassay was used due to the
amount of contaminants in saliva.
Displacement uses a micro capillary passage that immobilizes the antibodies to
the antigen of interest. As more antigen displaces the labeled antigen, the displaced
labeled antigen is detected.
Microfluidic
Electrophoresis
Capillary Electrophoresis (CE)7 uses a homogeneous phase immunoreaction,
which is normally very rapid due to mass transfer kinetics, followed by separation to
isolate and analyze the MMP-8 antigen. The unique fluid delivery capabilities of
microchip electrophoresis are necessary for automating immunoassays for use at the
point-of-care in the clinical environment. CE separates ionic species by their charge,
frictional forces, and hydrodynamic radius. Without CE, we would be unable to separate
the MMP-8 component from the rest of the saliva mixture.

6. The
Microchip
Electrophoretic
Immunoassay
(µCEI)
To include sample preparation and electrophoretic immunoassay on the same chip,
polymeric elements with certain physical patterns were photopatterned on class
microfluidic devices. The µCEI device consists of channels geared for specified
functions:
I. Sample Loading
II. Sample Enrichment
III. Rapid diffusive mixing of saliva with fluorescently labeled monoclonal antibody
[mAB] (MMP-8*)
IV. Subsequent Rapid Native Gel electrophoretic separation of MMP-8* from MMP-
8 complex.
Figure
1:
Multistep
Photopolymerization
of
µ CEI
Device
Fabrication
of
the
µ CEI
The three main regions fabricated were the size-exclusion membrane, a small pore-size
separation gel, and a larger pore-size loading gel.
Size-­‐Exclusion
Membrane
This portion was fabricated using laser photopolymerization of a solution of acrylamide
monomer, cross-linker, and photoinitiator using pressure-driven flow.

7. Pore-­‐Size
Separation
Gel
To define and localize the separation gel in the separation channel, all channels were
rinsed with a buffer and then pressure-loaded with the separation gel precursor solution.
UV photomasking was used to fabricate an intermediate porosity gel plug at the end of
the separation channel. Creating the plug resulted in a separation channel with separation
gel precursor and the elimination of bulk flow in the separation channel.
Pore-­‐Size
Loading
Gel
The loading gel was made using photopolymerization of an unmasked chip with a 100-W
UV lamp.
Layout
of
µ CEI
Chip
The µCEI device is labeled for
sample (S), buffer (B), sample waste
(SW), buffer waste (BW), and the
fluorescently labeled monoclonal
antibody to MMP-8 (mAB*). After
a buffer priming step, the mAB* is
loaded into the size-exclusion
membrane followed by the saliva
sample, both through the large pore-
size loading gel. Once the two
solutions are mixed, an electric
potential is applied across the
membrane so that enriched species
go into the separation channel and
start the electrophoretic
immunoassay. Later, the electric
potential is switched to take out the
membrane from the current path.
Figure
2:
Layout
of
µ CEI Chip

8. Quantifying
µ CEI
Assays
The sensitivity and dynamic range of µCEI assays allow us to vary the duration of
sample enrichment at the membrane or the magnitude of electric potential applied when
performing the enrichment step. Quantifying MMP-8 is the first step to moving away
from the binary nature of Point-of-Care clinical diagnostics and will help in monitoring
the disease activity in real time.
Macroscale
Comparison
of
Healthy
and
Periodontally
Diseased
Individuals.
While competitive immunoassay was used on the µCEI device, a regular colorimetric
sandwich ELISA was used in the macroscale to find the amount of concentration of
MMP-8 in saliva from the subjects. The severity of periodontal disease was assessed
through clinical examination, bleeding upon probing, pocket depth, and radiographic
bone loss. The most notable differences between healthy and diseased patients were in
the mean pocket depth and clinical attachment loss. A device capable of reporting
dynamic periodontal disease activity can also improve treatment by more effectively
timing the MMP inhibitor therapy since MMP-8’s active phase is correlated with
collagen deterioration.
Future
Directions
Researchers are motivated to achieve the potential of microfluidic immunoassays in
clinical diagnostics in order to take advantage of its miniaturization, integration, and
automation. However to do so, they must integrate the fields of material characterization,
fabrication, liquid transportation, surface modification, immobilization, and detection and
optimize them. The following are points to consider for the future development of
microfluidic immunoassays.

9. Mass
Production
for
Wide
Use
Although
PDMS
is
the
go-­‐to
polymer
for
microfluidic
research,
replicating
the
fabrication
process
takes
hours
of
time
that
would
limit
product
manufacturing.
In
order
to
make
massive
amounts
of
periodontal
disease
device
detectors,
other
techniques
for
should
be
produced
such
as
injection
molding
and
embossing.
Multiplexed
Assays
Single
chip
multiplexed
assays
are
an
important
feature
of
microfluidic
immunoassays.
There
have
been
recent
developments
for
a
suspension
array
for
a
multiplexed
immunoassay
with
Silica
Colloidal
Crystal
Beads
(SCCBs)8,9
that
show
different
reflective
spectra
as
colors.
Combining
microfluidic
devices
with
SCCBs
has
potential
for
clinical
applications
and,
regardless,
the
multiplexed
assay
will
remain
the
dominant
method
of
commercialization
for
microfluidic
immunoassays.
Surface
Modification
and
Immobilization
A
key
concern
for
immunoassays
is
the
nonspecific
adsorption
or
binding
to
molecules
instead
of
analytes,
which
affects
the
sensitivity
and
selectivity
of
the
assay.
The
competitive
immunoassay
is
a
good
alternative
for
impure
samples
and
the
advances
in
surface
chemistry
and
functional
modification
has
been
studied
extensively
enough
to
provide
a
solid
foundation
in
microfluidic
assays.
However
there
is
still
difficulty
in
surface
modification
and
immobilization
of
these
materials.
Purification
and
Concentration
As
mentioned
above,
the
complexity
and
small
amounts
of
antigens
in
samples
require
purification
and
concentration
procedures.
Microbeads
can
help
improve
sensitivity
and
helps
in
the
purification
process.
Their
increased
surface
area
and

10. ease
of
use
provide
a
promising
method
for
one-­‐step
purification
and
concentration
in
a
microfluidic
immunoassay.10
Detection
Compared
to
other
microcomponents,
detection
systems
for
immunoassays
are
bulky
and
expensive.
Although
some
integrated
detection
systems11
have
been
developed,
the
cost,
sensitivity,
and
fabrication
processes
restrict
their
practical
applications.
Thus,
developing
miniature,
portable,
and
inexpensive
detection
systems
with
an
acceptable
sensitivity
for
microfluidic
devices
are
in
great
demand.
Integration,
Packaging,
and
Price
Ultimately,
the
ideal
microfluidic
point
of
care
device
is
one
that
is
integrated,
dispable,
and
cheap.
Most
devices
released
are
used
by
trained
lab
personnel
and
other
auxiliary
machines
are
needed.
These
are
large
barriers
for
commercial
applications
but
an
integrated
low-­‐cost
microfluidic
immunoassays
with
multiplex
detection
function
is
possible,
with
further
research,
in
the
near
future.

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1
Microfluidic immunoassays as rapid saliva-based clinical diagnostics
Amy E. Herr†‡, Anson V. Hatch†, Daniel J. Throckmorton†, Huu M. Tran†, James S. Brennan†, William V. Giannobile§, and Anup
K. Singh†
†Biosystems Research Department, Sandia National Laboratories, Livermore, CA 94550; and §Michigan Center for Oral Research,
School of Dentistry, University of Michigan, Ann Arbor, MI 48106
Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved January 11, 2007 (received for review August 21,
2006)/5268–5273 ! PNAS ! March 27, 2007 ! vol. 104 ! no. 13
2
Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Scanometric DNA array de- tection with nanoparticle probes. Science 2000, 289(5485),
1757e1760.
3
“Smart” mobile affinity matrix for microfluidic immunoassays Noah Malmstadt, Allan S. Hoffman* and Patrick S. Stayton*
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA
Received 27th November 2003, Accepted 12th March 2004 First published as an Advance Article on the web 6th April 2004
Lab Chip, 2004, 4, 412–415
4
A. Hatch, A. E. Kamholz, K. R. Hawkins, M. S. Munson, E. A.
Schilling, B. H. Weigl and P. Yager, Nat. Biotechnol., 2001, 19,
461–465.
5
G. L. Lettieri, A. Dodge, G. Boer, N. F. de Rooij and E. Verpoorte, Lab
Chip, 2003, 3, 34–39.
6
D'Aiuto F, Parkar M, Andreou G, Suvan J, Brett PM, Ready D, Tonetti MS. (2004). Periodontitis and systemic inflammation: control
of the local infection is associated with a reduction in serum inflammatory markers. J Dent Res. 83(2):156-60.
7
Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline
determination
Nghia H. Chiem and D. Jed Harrison*, Clinical Chemistry 44:3 591–598 (1998)
8
Zhao, Y,; Zhao, X. W.; Sun, C.; Li, J.; Zhu, R.; Gu, Z. Z. Encoded silica colloidal crystal beads as supports for potential multiplex
immunoassay. Anal. Chem. 2008, 80(5), 1598e1605.
9
Sun, C.; Zhao, X. W.; Zhao, Y. J.; Zhu, R.; Gu, Z. Z. Fabrication of colloidal crystal beads by a drop-breaking technique and their
applica- tion as bioassays. Small 2008, 4(5), 592e596.
10
Matsunaga, T.; Maeda, Y.; Yoshino, T.; Takeyama, H.; Takahashi, M.; Ginya, H.; Aasahina, J.; Tajima, H. Fully automated
immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micro-polystyrene bead composites, ‘Beads on
Beads’. Anal. Chim. Acta 2007, 597(2), 331e339.
11
Hofmann, O.; Wang, X.; deMello, J. C.; Bradley, D. D. C.; deMello, A. J. Towards microalbuminuria determination on a disposable
diagnostic microchip with integrated fluorescence detection based on thin-film or- ganic light emitting diodes. Lab Chip 2005, 5(8),
863e868.