Utility of Centrifugation-Controlled Convective (C3) Flow for Rapid On-chip ELISA

Abstract

Miniaturizing the enzyme-linked immunosorbent assay (ELISA) protocols in microfluidics is sought after by researchers for a rapid, high throughput screening, on-site diagnosis, and ease in operation for detection and quantification of biomarkers. Herein, we report the use of the centrifugation-controlled convective (C3) flow as an alternative method in fluid flow control in a ring-structured channel for enhanced on-chip ELISA. A system that consists of a rotating heater stage and a microfluidic disk chip has been developed and demonstrated to detect IgA. The ring-structured channel was partially filled with microbeads (250 µm in diameter) carrying the capture antibodies and the analyte solution was driven by thermal convection flow (50 µL/min) to promote the reaction. The remaining part of the circular channel without microbeads served as the observation area to measure the absorbance value of the labeled protein. Currently, the system is capable of conducting four reactions in parallel and can be performed within 30 min at 300 G. A detection limit of 6.16 ng/mL using 24 µL of target sample (IgA) was observed. By simply changing the capture antibodies, the system is expected to be versatile for other immunoassays.

Figure 1

Fabricated microfluidic disk chip. (A) The assembled fluidic placed on the heater stage. Inset: microfluidic disk chip. (B) The disk consists of four layers of PDMS with PMMA bottom layer as support. (C) The ring channel served as the reaction chamber. Expanded image shows the height depression that prevents the (D) microbeads carrying the capture antibodies from escaping the main channel.

Figure 2

Developed rotating heater stage. (A) Actual set-up of the rotating stage with four separate pairs of heaters. (B) The high temp (orange) and the cold temp (blue) heaters were in contact with the conducting plates connected to separate heat sources. (C) Two pairs of heat sources were placed along the cavities of the fixed stage. (D) A stable temperature for the hot heater and the cold heater was observed for 15 min; 38 °C and 24 °C, respectively. Inset shows how the channel was aligned with the heaters.

Figure 3

Flow control in the ring-structured channel. (A) By centrifugation, the injected fluid was metered inside the ring-structured channel within 360 ms. (B) Illustration of the centrifugation-controlled convective (C3) flow inside the channel. (C) Observed convective flow under 300 G with a fixed thermal difference of 10 °C. (D) Flow rate inside the microchannel at various relative gravitational acceleration G at maximum and at minimum possible thermal difference in the set-up. Error bar represents standard deviation with n = 3.

Figure 4

Resulting simplified protocol for on-chip Enzyme-Linked Immunosorbent Assay (ELISA) using C3 flow. The U-shaped channel allows the easy exchange of the solution within the ring-structured channel by automatically metering the injected solution and flowing the excess (dashed line). The induced reflow with the ring-structured promotes a more efficient and rapid reaction.

Figure 5

Detection test using IgA. (A) Difference of the measurements based on convective flow and normal diffusion. Comparison of absorbance measurement using different amounts of fixed detection antibody on the microbeads and of enzyme-labeled antibody solution is also presented. The negative control of antigen sample was included to consider the effect of non-specific adsorption and determine the threshold. The highlighted region indicates the 1.66-fold signal enhancement. (B) Calibration curve of on-chip ELISA using C3 flow for IgA. Error bars represent standard deviation with n = 5. Visible difference of (C) 125 ng/mL and (D) blank, 0 ng/mL after adding the stop solution.