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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.

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 assays 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 (CEI) 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 crosscontamination 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 (CEI)

To include sample preparation and electrophoretic immunoassay on the same chip, polymeric elements with certain physical patterns were photopatterned on class microfluidic devices. The CEI 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 MMP8 complex.

Figure 1: Multistep Photopolymerization of CEI Device

Fabrication of the 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.

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 CEI Chip

The CEI 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 poresize 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 CEI Chip

Quantifying CEI Assays

The sensitivity and dynamic range of CEI 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 CEI 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-8s 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


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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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)/52685273 ! 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, 412415 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, 461465. 5 G. L. Lettieri, A. Dodge, G. Boer, N. F. de Rooij and E. Verpoorte, Lab Chip, 2003, 3, 3439. 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.

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 591598 (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.