Rapid on-chip ELISA aided by centrifugation-assisted thermal convection reflow

Abstract

Enzyme-linked immunosorbent assay (ELISA) is broadly used as a diagnostic tool in clinical applications for its precision, sensitivity, versatility, and quantifiability. Due to the sensitivity of the antibody-antigen interaction, it extends to a broad application in medical diagnosis, environmental measurement, and food safety evaluation. However, conventional ELISA protocols require multiple steps of adsorption, washing, and incubation of reagents. It's labor- intensive and time-consuming which often takes several hours, which can even last up to 2 days to perform a single assay. In addition, immunoagents used in ELISA are relatively expensive and usually done in a centralized lab with standard laboratory equipment. For health care administration, early and accurate detection is critical to increase survivability, improve disease management and treatment outcomes, and formulate preventive and personalized medicine. Thus, miniaturizing 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 disclose a system that consists of a rotating heater stage and a microfluidic disk chip that uses an alternative fluid flow control method in a ring-structured microchannel for enhanced on-chip ELISA. By simply altering the relative gravity acceleration, G, the thermal convective flow (Rayleigh−Bénard convection) can be controlled following the Boussinesq equation relation. This technique provides a reflow system which could significantly reduce the amount of reagents required to drive the flow and provide a more efficient mass transport for the immunoagent to migrate and conjugate to the detection surface. Currently, the system is capable of conducting four reactions in parallel and can be performed within 30 min at 300G. 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. In addition, the current sensitivity and detection limit could be further enhanced by adopting other techniques in minimizing the nonspecific binding on the surfaces or utilizing other materials aside from PDMS.