Automatic microfluidic enzyme-linked immunosorbent assay based on CLOCK-controlled autonomous centrifugal microfluidics

摘要

This paper reports on the development of an autonomous centrifugal microfluidic device, which automatically executes an enzyme-linked immunosorbent assay (ELISA) on a steady rotational frequency. The device was designed on the principles of CLOCK (Control of Liquid Operation on Centrifugal hydroKinetics) to achieve autonomous centrifugal microfluidics. These demonstrations confirmed that the device achieves accurate flow control of an automated ELISA with fluctuations of the interval time for implementation of each unit (antigen-antibody reaction, wash and injection of detection substrate) being less than 5%. The most optimal method for the immobilization of primary antibodies was investigated for the developed platform. We compared three methods of antibody immobilization on the developed platform, by using polystyrene microbeads, a polyurethane foam structure, and direct immobilization to the reaction chamber. We chose the use of polyurethane foam as the best immobilization method based on reaction efficiency and the convenient handling of the immobilized antibody on the solid phase. Measurement of human albumin was demonstrated using the developed device, and a calibration curve of human albumin detection was successfully prepared with an overall assay operation time of 18 min. The limit of detection (LOD) was estimated to be 0.516 ng/ml, which is comparable with the LOD of 0.707 ng/ml obtained by the assay on a conventional titer plate assay platform.