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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.
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.
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.
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
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.
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 µCEI Device
Fabrication of the µCEI 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.
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 µCEI 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 µCEI Chip
Quantifying µCEI 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.
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
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.