Influence of structural dimensions of micro-pillar array in reaction field on sensitivity of enzyme-linked immunosorbent assay (ELISA)

Abstract

For high sensitivity and rapid reaction of enzyme-linked immunosorbent assay (ELISA), the filmstack 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 mm) 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-mm and 50mm 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.

Rapid ELISA Using a Film-Stack Reaction Field with Micropillar Arrays

“…Figure 1 shows images of the film-stack reaction field. Film-stack reaction fields were fabricated by a nanoimprint process and a punch-press process for the molding of micropillars arrayed on base films, and the shaping and stacking of base films, respectively, in accordance with our previous studies [ 16 , 17 ]. A polyethylene terephthalate (PET) sheet and acrylic resin were used for the base film and micropillars, respectively.…”

“…IgA ELISA was carried out using wells with or without film-stack reaction fields with micropillar arrays having different dimensions at room temperature. The procedure followed is the same as described in our previous study [ 17 ]. For the incubation of human IgA (Bethyl Laboratories, Inc., Montgomery, TX, USA) on the surfaces of wells and the film-stack reaction fields, 100 µL of 1% purified human IgA diluted in 0.1 mol/L carbonate buffer solution (CBS, pH = 9.5) was added to each well.…”

“…To verify the effect of the film-stack reaction field with a micropillar array on the fluorescence intensity in terms of physical phenomena, a theoretical analysis of the physical parameters related with biomolecule transport was performed. This analysis is based on the computational simulation of its transport in a film-stack reaction field as shown in our previous study [ 17 ]. Figure 5 a,b shows the two-dimensional geometry models of the wells with and without the film-stack reaction field including the biomolecule transport in the theoretical analysis, respectively.…”

“…Furthermore, our previous study [ 17 ] showed the effect of the structural dimensions of a micropillar array in the film-stack reaction field on the fluorescence intensity of ELISAs by fluid-flow and particle-trajectory analyses by performing a computational simulation in addition to IgA ELISA tests for the optimized design of the film-stack reaction field. According to the computational simulation analysis in this previous study, the factors determining the fluorescence intensity in terms of the transport of biomolecules in the film-stack reaction field were identified, such as the surface area, the structural dimensions, the flow velocity, and the supplied number and diffusion distance of the biomolecules to the inertial space in the film-stack reaction field.…”

“…Furthermore, the dependence of the incubation time on the fluorescence intensity in the ELISA was investigated using this stirrer and the film-stack reaction field with micropillar arrays of different dimensions. Moreover, this study performed the theoretical modeling and analysis of the physical parameters related with the transport of biomolecules, such as the specific surface area, the diffusion time and distance, and the volumetric flow rate, in wells without and with film-stack reaction fields based on the computational analysis in our previous study [ 17 ] in light of the results of the ELISA tests for the optimization of the reaction-field design and the ELISA procedure with high sensitivity and a rapid reaction.…”