VEGF Detection via Simplified FLISA Using a 3D Microfluidic Disk Platform

Abstract

Fluorescence-linked immunosorbent assay (FLISA) is a commonly used, quantitative technique for detecting biochemical changes based on antigen-antibody binding reactions using a well-plate platform. As the manufacturing technology of microfluidic system evolves, FLISA can be implemented onto microfluidic disk platforms which allows the detection of trace biochemical reactions with high resolutions. Herein, we propose a novel microfluidic system comprising a disk with a three-dimensional incubation chamber, which can reduce the amount of the reagents to 1/10 and the required time for the entire process to less than an hour. The incubation process achieves an antigen-antibody binding reaction as well as the binding of fluorogenic substrates to target proteins. The FLISA protocol in the 3D incubation chamber necessitates performing the antibody-conjugated microbeads' movement during each step in order to ensure sufficient binding reactions. Vascular endothelial growth factor as concentration with ng mL^-1 is detected sequentially using a benchtop process employing this 3D microfluidic disk. The 3D microfluidic disk works without requiring manual intervention or additional procedures for liquid control. During the incubation process, microbead movement is controlled by centrifugal force from the rotating disk and the sedimentation by gravitational force at the tilted floor of the chamber.

Keywords: 3D microstructure; fluorescence-linked immunosorbent assay; lab-on-a-disk; vascular endothelial growth factor.

Figure 1

(a) Expanded view of the 3D microfluidic disk. (b) Image of the 3D microfluidic disk containing dyed water in the incubation (blue dye) and washing chambers (yellow dye). (c) Schematic of the cross-sectional illustrating the dimensions of the microfluidic chambers.

Figure 2

Schematic of the VEGF detection process via simplified microbead FLISA protocol. Biological assay between reagents is performed together in incubation step. The unbound dAb-fluorescent dye is eliminated in washing step. The fluorescence signal is measured for VEGF detection as much as the fluorescent dye attached to the microbead.

Figure 3

(a) Schematic of fluorescent dye-coupled VEGF on the microbeads. (b) The microbeads photographed with the longpass filter (>630 nm). (c) The dAb-bound fluorescent dye (green fluorescent protein) detected on the surface of the microbeads. (d) The merged image for the fluorescent dye and the microbeads.

Figure 4

(a–c) Green fluorescence intensity of the fluorescent dye-coupled VEGF on the microbeads after 2 h incubation, with reagent volumes of 100, 10, and 5 μL, respectively, and (d) 10 μL reagent volume for 1 h incubation.

Figure 5

A flow chart comparison of antigen detection techniques between microbead FLISA and traditional sandwich ELISA protocols.

Figure 6

Schematic of sectional view of the microfluidic components highlighting the sequential protocol of the simplified microbead FLISA; the processes indicated are (a) loading the wash buffer and reagents, (b) incubation, and (c) fluorescence detection, respectively.

Figure 7

(a) Bright-field and fluorescence images for fluorescent dye-coupled microbeads, with VEGF concentrations of 0 g mL⁻¹, 1 ng mL⁻¹, and 1 μg mL⁻¹ (scale bar = 100 μm). (b) Fluorescence intensity with varying VEGF concentrations.