RESUMO
A film-stacked structure consisting of polyethylene terephthalate (PET) films stacked in a gap of 20 µm that can be combined with 96-well microplates used in biochemical analysis has been developed by the authors. When this structure is inserted into a well and rotated, convection flow is generated in the narrow gaps between the films to enhance the chemical/bio reaction between the molecules. However, since the main component of the flow is a swirling flow, only a part of the solution circulates into the gaps, and reaction efficiency is not achieved as designed. In this study, an unsteady rotation is applied to promote the analyte transport into the gaps using the secondary flow generated on the surface of the rotating disk. Finite element analysis is used to evaluate the changes in flow and concentration distribution for each rotation operation and to optimize the rotation conditions. In addition, the molecular binding ratio for each rotation condition is evaluated. It is shown that the unsteady rotation accelerates the binding reaction of proteins in an ELISA (Enzyme Linked Immunosorbent Assay), a type of immunoassay.
RESUMO
Serum is commonly used as a specimen in immunoassays but the presence of heterophilic antibodies can potentially interfere with the test results. Previously, we have developed a microfluidic device called: 3D Stack for enzyme-linked immunosorbent assay (ELISA). However, its evaluation was limited to detection from a single protein solution. Here, we investigated the sensitivity of the 3D Stack in detecting a severe dengue biomarker-soluble CD163 (sCD163)-within the serum matrix. To determine potential interactions with serum matrix, a spike-and-recovery assay was performed, using 3D Stacks with and without surface modification by an EDC-NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) coupling. Without surface modification, a reduced analyte recovery in proportion to serum concentration was observed because of the Vroman effect, which resulted in competitive displacement of coated capture antibodies by serum proteins with stronger binding affinities. However, EDC-NHS coupling prevented antibody desorption and improved the sensitivity. Subsequent comparison of sCD163 detection using a 3D Stack with EDC-NHS coupling and conventional ELISA in dengue patients' sera revealed a high correlation (R = 0.9298, p < 0.0001) between the two detection platforms. Bland-Altman analysis further revealed insignificant systematic error between the mean differences of the two methods. These data suggest the potentials of the 3D Stack for further development as a detection platform.
