Bead-free digital enzyme-linked immunoassay on PDMS microwell array

Background

Proteins which are important biomarkers for disease diagnosis, prognosis and treatment are often present at very low concentrations in biological samples. Measuring these low-level proteins is important yet challenging. Conventional enzyme-linked immunoassays (ELISAs) usually lack the sensitive required. While ultrasensitive detection methods, such as digital ELISA have recently significantly advanced in sensitivity, they usually face high-cost of using paramagnetic beads or require complicated substrate fabrication, limiting their accessibility for large-scale screening or application in resource-limited settings. Therefore, there is a pressing need to develop cost-effective detection methods with ultrahigh sensitivity to accurately detecting low-level protein biomarkers.

Results

In the study, we developed a cost-effective and easily prepared PDMS microwell array platform for bead-free digital enzyme-linked immunoassay (ELISA) to achieve ultrasensitive protein detection. Both the immunocomplexes formation and separation process, which are two key steps in digital ELISA occur within the PDMS microwells, eliminating the need for paramagnetic beads and complex bead-separation systems. This approach significantly reduces detection costs compared to the widely used single-molecule arrays (SiMoAs)-based digital ELISA, which relies on large quantities of paramagnetic beads for immunocomplexes immobilization and intricate microwell array chips for immunocomplexes separation. Here, the PDMS microwell array chip was modified to generate large-scale femtoliter droplet arrays for single-molecule reactions, with minimized droplet evaporation and no cross-contamination. The PDMS microwell array platform was further successfully applied to single-molecule enzyme detection and cytokine analysis, achieving detection limits of 1.57 fM for streptavidin-β-galactosidase (SβG) and 12.3 fg/ml for human interleukin-6.

Significance

A low-cost PDMS microwell array platform for digital ELISA was developed. The fabrication of PDMS microwell array chip was straightforward by soft lithography, enabling mass production with small batch-to-batch variation. With its cost-effective fabrication process and high detection sensitivity, the PDMS microwell array would be a promising platform for ultrasensitive protein detection, especially in the area of early disease screening.