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 diseaseis a combination of multiple disease processes that share a common clinicalmanifestation. If not treated, it leads to tissue deterioration, loss of connective tissueattachment, and aleveolar bone loss. Furthering diagnostics research with microdevicescan eventually be used to frequently monitor episodic disease progression, enable earlydiagnosis of a disease, or continuously assess therapeutic efficacy. This paper uses microdevices to find matrix metalloproteinase-8 (MMP-8)1, amajor tissue-destructive enzyme in periodontal disease, in samples of saliva. To improvethe assay’s sensitivity to the enzyme, saliva pretreatment of mixing, incubation, andenrichment, was included before placing the solution in the quantitative immunoassay.The microchip electrophoretic immunoassay (µCEI) core of the device is based onphotolithographically fabricated molecular sieving gels to enrich the saliva sample andlater 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 touse, portable, user-friendly, and cheap. Colorimetric detection can fulfill theserequirments.2Immunoassays – Advantages Most biological procedures normally require solutions to be in an immobilized,biochemically active phase.3 Immobilization is key, especially for heterogeneousimmunoassays because it affects specificity and sensitivity. Switching from themacroscale to microscale depends on three main categories for biomolecularimmobilization: surface modification of microfluidic channel walls, packing microfluidicchannels with biomolecule-bearing beads, and packing microfluidic channels withbiomolecule-bearing porous slabs. For mircofluidic bioanalytical assays that do not usean immobilized phase, an assay based on the rate of diffusion of antibody-antigencomplexes4 in solution as well as a technique for maintaining beads in place in arecirculating flowstream without permanently immobilizing them is needed5.
3. Research on portable microfluidic devices for clinical diagnostics is a growingindustry because of its massive potential. These diagnostic devices would have lowermanufacturing costs, decreased sample size (here, a small amount of saliva is more thanenough), reproducible, and greater throughput. With the development of point-of-caremicrofluidic diagnostics, clients could perform more complex diagnoses in their ownhomes.Immunoassays – Disadvantages A significant disadvantage for microfluidic immobilization systems is its inherentirreversibility. A channel surface that has been chemically modified is difficult toremove, renew, or add an immobilized flexibility. This trait limits the flexibility of devicemanufacturing since each device must be made with a specific immobilized biochemistryfor a specific application. These devices also take longer to construct as they are morecomplex and the physics for macroscale machines differ from microscale devices due tothe 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 periodontalprobe. It is placed between the gums and the teeth and slipped about 2 to 3mm below thegum line. A subject with a peridontal pocket deeper than 7mm risks eventual tooth lossover the years. However, this disease could go on without recognition for many years.Types of Immunoassays Microarrays are commonly used to perform immunoassays. An immunoassaytypically immobilizes antibodies and exposes them to a biological sample. It is separatedinto four different types: direct-binding, sandwich (ELISA), competitive, anddisplacement. Direct-binding is when the antibody is labeled, normally fluorescently, and bindswith the target antigen. This method is not only quicker, but also avoids cross-contamination with a secondary antibody. However, direct-binding requires using everyantibody 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 andsecondary antibodies. The target antigen must have at least two sites to bind to theprimary and secondary antibody since both must act in the sandwich. This restrictssandwich assays to antigens with multiple binding sites for antibodies, such as proteins orpolysaccharides. However, sandwich is useful when there are low concentrations oftarget 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 signalsince fewer antibodies will be able to bind to the antigen in the well. The majoradvantage is that it can use crude or impure samples to selectively bind any antigenpresent. For the purposes of this paper, a competitive immunoassay was used due to theamount of contaminants in saliva. Displacement uses a micro capillary passage that immobilizes the antibodies tothe antigen of interest. As more antigen displaces the labeled antigen, the displacedlabeled 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 toisolate and analyze the MMP-8 antigen. The unique fluid delivery capabilities ofmicrochip electrophoresis are necessary for automating immunoassays for use at thepoint-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 separatethe 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 classmicrofluidic devices. The µCEI device consists of channels geared for specifiedfunctions: 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 DeviceFabrication of the µ CEI The three main regions fabricated were the size-exclusion membrane, a small pore-sizeseparation gel, and a larger pore-size loading gel.Size-‐Exclusion Membrane This portion was fabricated using laser photopolymerization of a solution of acrylamidemonomer, 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 wererinsed 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 ofthe separation channel. Creating the plug resulted in a separation channel with separationgel 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-WUV 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 ofsample enrichment at the membrane or the magnitude of electric potential applied whenperforming the enrichment step. Quantifying MMP-8 is the first step to moving awayfrom the binary nature of Point-of-Care clinical diagnostics and will help in monitoringthe disease activity in real time.Macroscale Comparison of Healthy and Periodontally Diseased Individuals. While competitive immunoassay was used on the µCEI device, a regular colorimetricsandwich ELISA was used in the macroscale to find the amount of concentration ofMMP-8 in saliva from the subjects. The severity of periodontal disease was assessedthrough clinical examination, bleeding upon probing, pocket depth, and radiographicbone loss. The most notable differences between healthy and diseased patients were inthe mean pocket depth and clinical attachment loss. A device capable of reportingdynamic periodontal disease activity can also improve treatment by more effectivelytiming the MMP inhibitor therapy since MMP-8’s active phase is correlated withcollagen deterioration.Future Directions Researchers are motivated to achieve the potential of microfluidic immunoassays inclinical diagnostics in order to take advantage of its miniaturization, integration, andautomation. However to do so, they must integrate the fields of material characterization,fabrication, liquid transportation, surface modification, immobilization, and detection andoptimize them. The following are points to consider for the future development ofmicrofluidic 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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