Abstract
ABSTRACTFor high sensitivity and rapid reaction of enzyme-linked immunosorbent assay (ELISA), the film-stack reaction field with micro-pillars array was designed and developed. The film-stack reaction field was fabricated by a nanoimprint process and an automatic punch-press process. The films with different gaps between micro-pillars (5, 10 and 50 μm) were prepared. These reaction fields were evaluated by IgA ELISA using 96-well microtitre plates and the computational simulation analysis of the fluid flow and the particle trajectory. Compared with ELISA using only the microtitre plate, higher detection sensitivity and shorter incubation time were achieved using the film-stack reaction field due to the increased surface area and the circulating flow through the space between films in a well by the rotation of the film-stack reaction field. Furthermore, in the ELISA results obtained using the film-stack reaction fields, the fluorescence intensities in 10-μm and 50-μm pillar gaps were the minimum and maximum values, respectively. This trend was due to the flow rate between micro-pillars, and the number and the diffusion distance of supplied biomolecules to the inertial space in the film-stack reaction field. In simulation results, the trend of the number of adsorbed biomolecule particles with different gaps between micro-pillars was in agreement with the trend in the ELISA results. Hence, these simulation analyses were validated in the quantitative evaluation of this reaction field and could be applied in the design of this reaction field as an effective design tool.
Highlights
Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are very important methods for the detection of various proteins in immunologic reactions with an application in numerous disciplines including the diagnosis of infectious diseases [1,2,3,4,5,6]
Considering the above factors, the optimized design of the film-stack reaction field with the analyses of the fluid flow and the particle trajectory can be expected to produce a reaction field with high sensitivity and rapid reaction
These reaction fields were characterized by IgA ELISA, the fluid-flow analysis based on finite elements method (FEM) and the particle trajectory analysis using the fluid-flow analysis
Summary
Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are very important methods for the detection of various proteins in immunologic reactions with an application in numerous disciplines including the diagnosis of infectious diseases [1,2,3,4,5,6]. With the development of the microfabrication techniques, micro bio-analysis devices (MBDs), such as micro-total analysis systems and lab-on-a-chip, are demanded to achieve rapid detection of biomolecules [10,11,12]. This is due to the short diffusion distance of biomolecules and the high surface-to-volume ratio by miniaturizing a reaction field. The top-down process reduces a large piece of material to produce the form with a desired shape and size of micrometer scale It has multiple steps which induce high cost for mass production. The design parameters, such as the size, shape, surface area and surface properties of the reaction field, are important factors because
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